Differentiation of primate pluripotent stem cells to hematopoietic lineage cells

ABSTRACT

The invention provides methods of differentiating primate pluripotent stem cells into cells of hematopoietic lineage. The invention further provides hematopoietic lineage cells differentiated from primate pluripotent stem cells, as well as methods of using the same and kits comprising the same.

This application claims priority to provisional application No.61/039,835, filed Mar. 27, 2008 and provisional application No.61/081,242, filed Jul. 16, 2008, both of which are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of stem cell biology.

BACKGROUND

Pluripotent stem cells have the ability to both proliferate continuouslyin culture and, under appropriate growth conditions, differentiate intolineage restricted cell types representative of all three primary germlayers: endoderm, mesoderm and ectoderm (U.S. Pat. Nos. 5,843,780;6,200,806; 7,029,913; Shamblott et al., (1998) Proc. Natl. Acad. Sci.USA 95:13726; Takahashi et al., (2007) Cell 131(5):861; Yu et al.,(2007) Science 318:5858). Defining appropriate growth conditions forparticular lineage restricted cell types will provide virtually anunlimited supply of that cell type for use in research and therapeuticapplications.

It would be particularly useful to be able to differentiate pluripotentstem cells into hematopoietic lineage cells. Hematopoietic lineage cellsdevelop from the mesoderm layer and include both white and red bloodcells, which constitute the immune and circulatory systems,respectively. An unlimited supply of these cells would provide the toolsnecessary to more fully understand both the development and functioningof both the immune and circulatory systems. It would also provideinsight into strategies for modulating immune responses, both beneficialand harmful.

The immune system provides for an innate or non-specific immune responseas well as an adaptive or specific immune response. The adaptive immuneresponse is a long lasting protective response and it is this responsemost vaccine protocols seek to stimulate. Cellular participants in theadaptive immune response include lymphocytes (T cells and B cells) aswell as dendritic cells (DC). T cells and B cells eliminate targetpathogens by specifically recognizing antigenic epitopes expressed onthe pathogen. T cells have cytotoxic capability that is especially adeptat targeting virally infected and tumor cells. B cells secreteantibodies which bind target antigens and activate the complement systemfacilitating opsonization and lysis of the target. Both responses arecharacterized as memory responses and thus are protective over a periodof time. DC play an important role in initiating the adaptive immuneresponse. They present antigen to the lymphocytes in the context of theappropriate major histocompatibility complex (MHC) and thus provide theinitial stimulus for mounting the adaptive immune response. A readysupply of DC could provide a means for generating either a therapeuticor prophylactic immune response in a host.

A number of studies have demonstrated the potential of DC as vehiclesfor generating an adaptive immune response (see, e.g., Mayordomo et al.,(1995) Nature Med 1:1297; Celluzi et al., (1996) J. Exp. Med. 183:283;Su et al., (1998) J. Exp. Med. 188:809) including studies that haveinvestigated the effects of irradiating DC (see, e.g., Cao et al. (2004)Cell Biology International 28:223; Merrick et al., (2005) BritishJournal Of Cancer 92:1450; Young et al. (1993) Blood 81(11):2987;Denfield et al. (2001) Journal Of Leukocyte Biology 69:548; Dudda et al.(2004) Journal of Investigative Dermatology 122:945).

The potential of dendritic cells along with the promise of pluripotentstem cells have lead several investigators to attempt to differentiatepluripotent stem cells into DC or their precursors (see, e.g. U.S. Pat.No. 7,247,480; U.S. Patent Publication Nos.: 2002/0086005; 2003/0153082;2006/0275901; 2006/0147432; 2006/0063255; 2006/0147432; Fairchild etal., (2005) International Immunopharmacology 5:13; Tacken et al., (2007)Nature Reviews Immunology 7:790; Senju et al., (2007) Stem Cells25(11):2720; Sluvkin et al., (2006) J of Immunology 176:2924; Li et al.,(2001) Blood 98(2):335; Kaufman et al., (2001) Proc Natl Acad Sci98(19):10716; Chadwick et al., (2003) Blood 102(3):906; Zhan et al.,(2004) Lancet 364:163; Fairchild et al., (2000) Current Biology 10:1515;Kennedy et al., (2007) Blood 109(7):2679; Ng et al., (2005) Blood106(5):1601; Fehling et al., (2003) Development 130:4217; Lu et al.,(2004) Blood 103(11):4134; Zambidis et al., (2005) Blood 106(3):860;Bandi et al., (2008) AIDS Research and Therapy 5:1; Pick et al., (2007)Stem Cells 25:2206).

Many of these investigators relied on stromal cells and/or feeder cellsto grow and/or differentiate their stem cells. The use of feeder cellsand stromal cells is cumbersome, expensive, time consuming and difficultto scale up. Some of these investigators used animal products such asanimal serum in their protocols. Using animal products, however, carrieswith it the risk of contamination of the cells with zoogenic infectiousagents. Still other investigators relied on random or poorly formulateddifferentiation protocols resulting in unpredictable outcomes andgenerally low yield of product.

There is a need for hematopoietic lineage cells differentiated frompluripotent stem cells and for methods of producing these cells that isscalable, economical, efficient, reliable, safe, and capable ofproviding good yield of product. Various embodiments of the inventiondescribed herein meet these needs and other needs as well.

SUMMARY OF THE INVENTION

In certain embodiments the invention provides for the in vitrodifferentiation of primate pluripotent stem cells (pPS) intohematopoietic lineage cells. The pPS cells may be human pluripotent stemcells that are suitable for differentiation into human hematopoieticlineage cells. The hematopoietic lineage cell may include, for example,an immature dendritic cell (imDC), a mature dendritic cell (mDC), amyeloid precursor cell, a monocyte.

In certain other embodiments the invention provides a method for the invitro differentiation of pPS cells into mesoderm cells.

Differentiation of pPS cells into hematopoietic lineage cells mayinclude contacting cells in vitro, e.g. pPS cells, with adifferentiation cocktail comprising a plurality of exogenous cytokines,and/or a plurality of exogenous ligands to proteins expressed on thecell surface (including, for example, exogenous ligands to cytokinereceptors such as an antibody which specifically binds to the cytokinereceptor), such that the cell population differentiates into a cellhaving a different phenotype, e.g. the phenotype of a hematopoieticlineage cell, while maintaining essentially the same genotype. Suitableexogenous cytokines may include a plurality of the following:granulocyte-macrophage colony stimulating factor (GM-CSF), bonemorphogenic protein 4 (BMP-4), vascular endothelial growth factor(VEGF), stem cell factor (SCF), thrombopoietin (TPO), fetal liver kinaseligand (FLT3L), interleukin 4 (IL-4) and interleukin 3 (IL-3).

Reference to the cells having the same genotype is not intended to implythat the cells cannot be genetically manipulated by the human hand(embodiments encompassing genetically altered cells are describedinfra), or that very minor changes (e.g., less than a fraction of apercent of the entire genome) might occur spontaneously (e.g. in thenon-coding regions), but rather merely to suggest that the act ofdifferentiating the cells from pPS cells into cells of hematopoieticlineage will not, by itself, result in an altered genotype. Typicallythe genetic identity between a parental (undifferentiated cell) and itsdifferentiated progeny will be similar to the genetic identity foundbetween identical twins.

In certain embodiments the method of in vitro differentiation of pPScells into hematopoietic lineage cells may be practiced serum free. Insome embodiments the method of differentiation of pPS cells intohematopoietic lineage cells may be practiced feeder free. In variousembodiments the method of differentiation of pPS cells intohematopoietic lineage cells may be practiced stromal cell free. Incertain embodiments the method of differentiation of pPS cells intohematopoietic lineage cells may be practiced without the addition ofexogenous IL-3 or the addition of an exogenous ligand to the IL-3receptor.

In some embodiments the invention provides a method of differentiatingpPS cells in vitro into imDC comprising contacting the pPS cells with aplurality of exogenous cytokines comprising GM-CSF and BMP-4. Theplurality of exogenous cytokines may further include one or more of thefollowing: VEGF, SCF, TPO, FLT3L, and IL-3. In some embodiments IL-4 mayalso be included in this differentiation cocktail.

In yet other embodiments the invention provides a method ofdifferentiating pPS cells in vitro into mDC comprising 1) contacting thepPS cells with a differentiation cocktail comprising a plurality ofexogenous cytokines, and/or a plurality of exogenous ligands to aprotein expressed on the cell surface, e.g., to a cytokine receptor,suitable for differentiating pPS cells to imDC thereby differentiatingpPS cells into imDC; and 2) contacting the imDC with a maturationcocktail comprising a plurality of exogenous cytokines, and/or exogenousligands to a protein expressed on the cell surface, e.g., to a cytokinereceptor, suitable for facilitating the maturation of the imDC to mDCthereby differentiating the imDC into mDC. The differentiation cocktailmay comprise a plurality of the following: GM-CSF, VEGF, BMP-4, SCF,TPO, FLT3L and IL-3. The maturation cocktail may comprise a plurality ofthe following: tumor necrosis factor α (TNFα), interleukin 1β (IL1β),interferon γ (IFNγ), prostaglandin E2 (PGE2), polyinosinic:polycytidylic acid (POLY I:C), interferon α (IFNα), CD40L and GM-CSF.

In one embodiment the invention provides a method of differentiating pPScells in vitro into mDC comprising contacting the pPS cells with adifferentiation cocktail comprising BMP-4, GM-CSF, VEGF and SCF and asuitable maturation cocktail, e.g., a maturation cocktail comprisingGM-CSF, IFNγ, TNFα, IL1β, and PGE2. In this embodiment IL-4 may also beincluded in the differentiation cocktail.

In some embodiments the composition of the differentiation cocktail maystay the same over the course of the differentiation of pPS cells tohematopoietic lineage cells. For example the differentiation cocktailmay comprise BMP-4, GM-CSF, VEGF and SCF through out the course ofdifferentiating the pPS cells to imDC. In some embodiments IL-4 may alsobe included in the differentiation cocktail.

In other embodiments the composition of the differentiation cocktail maychange over the course of the differentiation protocol. Thus in someembodiments of the invention the differentiation cocktail may comprise 4exogenous cytokines, or 4 exogenous ligands to cell surface proteins forone or more steps of the protocol while in other steps of thedifferentiation protocol the differentiation cocktail may comprise 3, 2,or 1 exogenous cytokine(s) or exogenous ligand(s) to a cell surfaceprotein. For example the cells may be contacted first with adifferentiation cocktail comprising BMP-4, VEGF and SCF (GM-CSF mayoptionally be included in this first step), followed by adifferentiation cocktail comprising VEGF, SCF and GM-SCF, followed by adifferentiation cocktail comprising SCF and GM-CSF, followed by adifferentiation cocktail comprising GM-CSF, followed by adifferentiation cocktail comprising GM-CSF and interleukin 4 (IL-4),thus differentiating pPS cells into imDC. The imDC may then be contactedwith a suitable maturation cocktail, e.g., a maturation cocktailcomprising IFNγ, TNFα, IL1β, and PGE2.

In still further embodiments the invention provides a method ofdifferentiating pPS cells in vitro into cells expressing one or more ofthe following: CD83, CD14, MHC I, MHC II, CD11c and CD11b comprisingcontacting the pPS cells with a plurality of the following: GM-CSF,BMP-4, VEGF, SCF, FLT3L, TPO, and IL-3 and/or exogenous ligands to acell surface protein.

In yet further embodiments the invention provides a method ofdifferentiating in vitro a cell expressing stage specific embryonicantigen 3 (SSEA3), stage specific embryonic antigen 4 (SSEA4) andmarkers detectable using antibodies designated Tra-1-60, and Tra-1-81into cells expressing one or more of the following: CD83, CD14, MHC I,MHC II, CD11c and CD11b comprising contacting the pPS cells with aplurality of the following: GM-CSF, BMP-4, VEGF, SCF, FLT3L, TPO, andIL-3 and/or exogenous ligands to a cell surface protein.

In still further embodiments the invention provides a method ofdifferentiating pPS cells in vitro into cells expressing CD83 CD14, MHCI, MHC II, CD11c and CD11b comprising contacting the pPS cells with aplurality of exogenous cytokines comprising GM-CSF and BMP-4 and/or anexogenous ligand to a cell surface receptor. The plurality of exogenouscytokines may further include one or more of the following: VEGF, SCF,FLT3L, TPO, and IL-3 and/or exogenous ligands to a cell surface protein.Examples of cell surface proteins may include a receptor for one of thepreviously mentioned cytokines.

In some embodiments the invention provides a method of differentiatingpPS cells in vitro into cells expressing one or more of the following:MHC-I, MHC-II, CD83, CD205, CD11b, CCR7, CD40, CD86, CD123, CD11ccomprising contacting the pPS cells with 1) a differentiation cocktailand then contacting the cells of 1) with a maturation cocktail. Thedifferentiation cocktail may comprise a plurality of the following:GM-CSF, BMP-4 VEGF, SCF, FLT3L, TPO, IL-4 and IL-3 and/or exogenousligands to a cell surface protein. The maturation cocktail may comprisea plurality of the following: GM-CSF, TNFα, IL1β, IFNγ, PGE2, POLY I:C,IFNα and/or exogenous ligands to a cell surface protein. Examples ofcell surface proteins may include a receptor for one of the previouslymentioned cytokines.

In certain embodiments the invention provides a method ofdifferentiating pPS cells in vitro into cells expressing CD83 comprisingcontacting the pPS cells with a differentiation cocktail and amaturation cocktail. The differentiation cocktail may comprise GM-CSFand BMP-4 and/or an exogenous ligand to a cell surface receptor. In someembodiments the differentiation cocktail may further include one or moreof the following: VEGF, SCF, FLT3L, TPO, IL-4 and IL-3. The maturationcocktail may comprise a plurality of the following: TNFα, IL1β, PGE2,POLY I:C, IFNα, CD40L and GM-CSF. In some embodiments the cellexpressing CD83 may also express one or more of the following CD86,CD14, CD11b, CD11c, CD205, MHC I and MHC II. In some embodiments thedifferentiation cocktail may comprise exogenous ligands to a cellsurface protein, such as a cytokine receptor.

In still other embodiments the invention provides a method ofdifferentiating pPS cells in vitro into a population of cells expressingCD45 and CD11c comprising contacting the pPS cells with a plurality ofexogenous cytokines comprising GM-CSF and BMP-4 and/or an exogenousligand to a cell surface receptor. In some embodiments the plurality ofexogenous cytokines may further include one or more of the following:VEGF, SCF, FLT3L, TPO, and IL-3 and/or exogenous ligands to a cellsurface protein. The CD45 expressing cells maybe CD45^(hi) cells.

In further embodiments the invention provides a method ofdifferentiating in vitro a cell expressing stage specific embryonicantigen 3 (SSEA3), stage specific embryonic antigen 4 (SSEA4) andmarkers detectable using antibodies designated Tra-1-60, and Tra-1-81into into a population of cells expressing CD45 and CD11c comprisingcontacting the cell expressing stage specific embryonic antigen 3(SSEA3), stage specific embryonic antigen 4 (SSEA4) and markersdetectable using antibodies designated Tra-1-60, and Tra-1-81 with aplurality of exogenous cytokines comprising GM-CSF and BMP-4 and/or anexogenous ligand to a cell surface receptor. In some embodiments theplurality of exogenous cytokines may further include one or more of thefollowing: VEGF, SCF, FLT3L, TPO, and IL-3 and/or exogenous ligands to acell surface protein. The CD45 expressing cells maybe CD45^(hi) cells.

Reference to differentiating cells expressing one or more markers mayinclude embodiments where expression of the referenced marker isincreased (e.g. as a result of the differentiation) when compared to astarting cell population (e.g. a precursor cell population with respectto the differentiated cell population).

In further embodiments the invention provides a method ofdifferentiating pPS cells in vitro into mesoderm comprising contactingthe pPS cells with a differentiation cocktail comprising a plurality ofexogenous cytokines. The differentiation cocktail may include aplurality of the following: BMP-4, VEGF, SCF, FLT3L and GM-CSF and/orexogenous ligands to a cell surface protein. In one embodiment thedifferentiation cocktail may comprise BMP-4, VEGF, SCF.

In other embodiments the invention provides a cell culture comprising afirst population of cells comprising pPS cells and second population ofcells comprising a hematopoietic lineage cell. Hematopoietic lineagecells may include one or more of the following: hemangioblasts,hematopoietic stem cells, myeloid progenitor cells, granulomonocyticprogenitor cells, monocytes, imDC and mDC. In some embodiments the cellculture may comprise a plurality of exogenous cytokines and/or ligandsto cell surface proteins such as cytokine receptors. Suitable exogenouscytokines may include the following: GM-CSF, VEGF, BMP-4, SCF, FLT3L,IL-4, TPO, TNFα, IL1β, IFNγ, PGE2, POLY I:C, IFNα. The cell culture mayalso comprise exogenous CD40L. In some embodiments the cell culture mayoptionally not comprise exogenous IL-3 or an exogenous ligand to theIL-3 receptor. In some embodiments the cell culture may be feeder free.In some embodiments the cell culture may be stromal cell free. In someembodiments the cell culture may be serum free.

In yet other embodiments the invention provides a cell culturecomprising a first population of cells comprising pPS cells and secondpopulation of cells comprising a DC, e.g., an mDC, an imDC. In someembodiments the cell culture may comprise a plurality of exogenouscytokines. Suitable exogenous cytokines may include the following:GM-CSF, VEGF, BMP-4, SCF, TPO, TNFα, FLT3L, IL1β, IL-4, IFNγ, PGE2, POLYI:C, IFNα. The cell culture may also comprise exogenous CD40L. In oneembodiment the invention provides a cell culture comprising a firstpopulation of cells comprising pPS cells and second population of cellscomprising a DC, e.g., an mDC, an imDC and exogenous BMP-4 and GM-CSF.In some embodiments the cell culture may optionally not compriseexogenous IL-3 or an exogenous ligand to the IL-3 receptor. In someembodiments the cell culture may be feeder free. In some embodiments thecell culture may be stromal cell free. In various embodiments the cellculture may be serum free. In certain embodiments the cell culture maybe irradiated. For example, an irradiated cell culture may include acell culture comprising mDC. The irradiated cell culture may alsocomprise at least one pPS cell. In other embodiments the cells may becontacted with a chemical agent suitable for inhibiting cell divisionsuch as a chemotherapeutic, e.g., mitomycin, cisplatin.

In further embodiments the invention provides a method of inhibitingcell division in a cell culture comprising contacting the cell culturewith a source of radiation or a chemical agent, wherein the cell culturecomprises at least one pPS cell and mDC differentiated in vitro from pPScells.

In still other embodiments the invention provides a method of making animmuno-modulating preparation comprising 1) differentiating at least aportion of a population of pPS cells into mDC cells thereby obtaining amixed population of cells comprising mDC and at least one pPS cell and2) contacting the mixed population of cells of 1) with a radiationsource or a chemical agent thereby obtaining an immuno-modulatingpreparation. The method may further comprise contacting the mixedpopulation of cells comprising mDC with an antigen, e.g. a protein or apeptide. The population of cells may be contacted with an antigen beforethe cells are contacted with the radiation. The immuno-modulatingpreparation may stimulate an immune response to an antigen.

In further embodiments the invention provides a method of making animmuno-modulating preparation comprising 1) differentiating at least aportion of a population of pPS cells into a population comprising imDCcells thereby obtaining a mixed population of cells comprising imDC andat least one pPS cell; 2) contacting the population of cells comprisingimDC with a nucleic acid encoding an antigen; 3) contacting thepopulation of cells of 2) with a maturation cocktail such that the imDCmature into mDC wherein the population comprises at least one pPS celland 4) contacting the mixed population of cells of 3) with a radiationsource or a chemical agent thereby obtaining an immuno-modulatingpreparation. The immuno-modulating preparation may stimulate an immuneresponse to antigen.

In yet other embodiments the invention provides a method of making animmuno-modulating preparation comprising 1) differentiating at least aportion of a population of pPS cells into a population comprising imDCcells thereby obtaining a mixed population of cells comprising mDC andat least one pPS cell; 2) contacting the population of cells of 1) witha maturation cocktail such that the imDC mature into mDC, wherein thepopulation of cells comprises at least one pPS cell 3) contacting thepopulation of cells comprising mDC with a nucleic acid encoding anantigen; and 4) contacting the mixed population of cells of 3) with aradiation source or a chemical agent thereby obtaining animmuno-modulating preparation. The immuno-modulating preparation maystimulate an immune response to antigen.

In still other embodiments the invention provides an immuno-modulatingcomposition comprising a mitotically inactivated mDC which is the invitro progeny of a pPS cell. The composition may be irradiated ortreated with a chemical agent suitable for inhibiting cell division suchas a chemotherapeutic, e.g. mitomycin, cisplatin in order to mitoticallyinactivate the cells. In some embodiments the immuno-modulatingcomposition may comprise a DC e.g. an mDC or an imDC contacted with anantigen or a nucleic acid encoding an antigen prior to irradiation. Theimmuno-modulating response may be one that stimulates an immune responseto an antigen.

In other embodiments the invention provides a method of stimulating animmune response to an antigen comprising a) obtaining an mDCdifferentiated in vitro from a pPS cell; b) contacting the mDC with anantigen or a nucleic acid molecule that encodes an antigen; c)contacting the mDC of b) with a radiation source or a chemical agentsuitable for inhibiting cell division, e.g. mitomycin; d) contacting themDC of c) with an immunologically competent cell thereby stimulating animmune response to the antigen.

In other embodiments the invention provides a method of stimulating animmune response to an antigen comprising a) obtaining an imDCdifferentiated in vitro from a pPS cell; b) contacting the imDC with anucleic acid molecule that encodes an antigen; c) contacting the imDCwith a maturation cocktail (as described herein) such that the imDCmatures into an mDC d) contacting the mDC of c) with a radiation sourceor a chemical agent suitable for inhibiting cell division, e.g.mitomycin; e) contacting the mDC of d) with an immunologically competentcell thereby stimulating an immune response to the antigen.

In other embodiments the invention provides a method of stimulating animmune response to an antigen comprising a) contacting a pPS cell with adifferentiation cocktail and a maturation cocktail such that the pPSdifferentiates into an mDC; b) contacting the mDC of a) with an antigenor a nucleic acid molecule that encodes an antigen; c) contacting themDC of b) with an immunologically competent cell thereby stimulating animmune response to the antigen. The differentiation cocktail maycomprise a plurality of exogenous cytokines and/or a plurality ofexogenous ligands to cell surface proteins. The differentiation cocktailmay comprise GM-CSF, BMP-4, VEGF, SCF, FLT3L, TPO, IL-4 and IL-3. Thematuration cocktail may comprise one or more of the following: GM-CSF,TNF-α, IL1β, IFNγ, PGE2, POLY I:C, IFNα. In some embodiments the cellculture may optionally not comprise exogenous IL-3 or an exogenousligand to the IL-3 receptor. In some embodiments the cell culture may befeeder free. In some embodiments the cell culture may be stromal cellfree. In some embodiments the cell culture may be serum free.

In still other embodiments the invention provides a kit for stimulatingan immune response to an antigen comprising 1) a cell culture comprisingpPS cells and DC and 2) one or more containers. The DC may be mDC orimDC. The cell culture may comprise exogenous cytokines and/or orexogenous ligands to cell surface proteins. The exogenous cytokinesand/or or exogenous ligands to cell surface proteins may include aplurality of the following: GM-CSF, VEGF, BMP-4, SCF, FLT3L, TPO, IL-4,IL-3, TNFα, IL1β, IFNγ, PGE2, POLYI:C, IFNα, CD40L. In some embodimentsthe cell culture may optionally not comprise exogenous IL-3 or anexogenous ligand to the IL-3 receptor. In some embodiments the cellculture may be feeder free. In some embodiments the cell culture may bestromal cell free. In some embodiments the cell culture may be serumfree.

In yet other embodiments the invention provides a kit for stimulating animmune response to an antigen comprising 1) an irradiated mDC which isthe in vitro progeny of a pPS cell and 2) one or more containers.

In still other embodiments the provides a kit for stimulating an immuneresponse to an antigen comprising 1) a mitotically inactivated mDC whichis the vitro progeny of a pPS cell; and 2) one or more containers. ThemDC may be contacted with a chemical agent suitable for inhibiting celldivision to mitotically inactivate the cells. A suitable chemical forinhibiting cell division may include mitomycins such as mitomycin C.Alternatively the mDC may be contacted with a radiation source tomitotically inactivate the cells.

In further embodiments the invention provides a system for theproduction of mitotically inactive antigen presenting cells comprisinga) a first isolated cell population comprising pPS cells and b) a secondisolated cell population comprising mitotically inactivated maturedendritic cells which are the in vitro progeny of a portion of the pPScells. The mature dendritic cells may be mitotically inactivated byirradiation or by contact with a chemical agent. It is contemplated thatthe first isolated cell population comprising the pPS cells (e.g. theportion not used to make the mDC) may be used to make more of the secondisolated population by differentiating the first population of cells invitro.

It is contemplated that any of the embodiments of the invention may bepracticed by substituting one or more of the following sub-groupings ofpPS cells: human embryonic stem cells, human embryonic germ cells,rhesus stem cells, marmoset stem cells, nuclear transfer stem cellsand/or induced pluripotent stem cells, all of which are described infra.

DESCRIPTION OF THE FIGURES

FIG. 1A provides a schematic diagram of one differentiation protocolused for differentiating pPS cells to mDC.

FIG. 1B is a photograph of a light microscopy image of hES cells grownin X-VIVO™ 10 media.

FIG. 1C is a flow cytometric histogram showing the expression level ofvarious markers found on undifferentiated hES.

FIG. 2A and FIG. 2B are photomicrographs of embryoid bodies (FIG. 2B)and progenitor cells (FIG. 2A).

FIGS. 3A and 3B are graphs showing expression of various transcriptionfactors over time in a cell culture undergoing differentiation.

FIG. 3C shows expression of CD34 and CD45 over time as measured by flowcytometry in a cell culture undergoing differentiation.

FIG. 3D is a photomicrograph of a cystic embryoid body.

FIG. 3E shows expression of CD13 and CD14 over time as measured by flowcytometry in a cell culture undergoing differentiation.

FIG. 3F shows expression of CD14 in both CD45^(hi) population (top 2panels) and CD45^(lo) population (lower two panels) as measured by flowcytometry.

FIG. 3G shows expression of CD11c, CD11b, CD83, CD86 (bottom twopanels), HLA-I and HLA-II (top right panel) in a CD45^(hi) population asmeasured by flow cytometry. The top left panel shows gating of theCD45hi and lo populations.

FIG. 4A shows a flow cytometric histogram analysis of markers for imDC.

FIG. 4B shows a flow cytometric histogram analysis of markers for mDC.

FIG. 4C is a graph showing transcription factor expression indifferentiating cell cultures over time.

FIG. 4D is a photomicrograph of a DC cluster.

FIG. 4E is a photomicrograph of DC stained with May Grunwald stain.

FIG. 5A shows gating of cells by flow cytometry for dendritic cells (R1)(top panel) and demonstrates that the cell population gated fordendritic cells can take up and proteolytically process the modelantigen DQ-OVA (lower panel).

FIG. 5B is a graph showing that DC can process and present mumps antigento induce IFNγ production by T lymphocytes.

FIG. 6A, FIG. 6B and FIG. 6C are graphs comparing the cytokine profileof imDC and mDC.

FIG. 6D is a graph showing DC migration in response to MIP3β.

FIG. 7A is a graph showing that mDC can stimulate allogeneic cells in amixed lymphocyte reaction (MLR).

FIG. 7B is a graph showing stimulation of IFN-y secretion by effector Tcells m response to a CMV peptide antigen presented on HLA-A2 by mDC(ES-DC).

FIG. 7C shows a flow cytometry analysis of CFSE labeled T lymphocyteproliferation in response to a CMV peptide presented on HLA-A2 by mDC(ES-DC).

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D are graphs showing stimulation ofIFNγ secretion by effector T cells in response to an hTERT peptideantigen presented on HLA-A2 by mDC (hES-DC).

FIG. 9A, FIG. 9B, and FIG. 9C show flow cytometry analysis of CFSElabeled T lymphocyte proliferation in response to an hTERT peptideantigen presented on HLA-A2 by mDC.

FIG. 10 is a graph comparing the stimulation of an antigen specific Tcell response by irradiated mDC (hES-DC) versus non-irradiated mDC(hES-DC) either pulsed or unpulsed with peptide antigen.

FIG. 11 is a graph comparing DC migration in response to chemotacticligand MIP3 of irradiated mDC (hES-DC) versus non-irradiated mDC(hES-DC).

FIG. 12 is graph comparing mDC yields in hES cells grown in eitherXVivo10™ or mTeSR™ media.

FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D are graphs comparing surfacemarker expression of DC differentiated in vitro from hES cells andmatured in either Cellgro™ or X Vivo-15™ media.

FIG. 14 is a graph comparing cell migration of hES derived mDC culturedin Cellgro™ or X-Vivo-15™.

FIG. 15 is a graph comparing IL-12 production from hESC derived DCcultured in either Cellgro™ or X-Vivo-15™ media either with or withoutthe addition of exogenous IL-4 to the maturation cocktail.

FIG. 16 is graph comparing IFNγ production from TERT specific T cellsco-incubated with mDCs transfected with GFP; mDCs transfected withhTERT-LAMP and T cells alone (without co-incubation with mDC cells).

DEFINITIONS

About, as used herein to refer to an amount or a value means + or −5% ofstated amount or value.

Cell culture, as used herein, refers to a plurality of cells grown invitro over time. The cell culture may originate from a plurality of pPScells or from a single pPS cell and may include all of the progeny ofthe originating cell or cells, regardless of 1) the number of passagesor divisions the cell culture undergoes over the lifetime of theculture; and 2) any changes in phenotype to one or more cells within theculture over the lifetime of the culture (e.g. resulting fromdifferentiation of one or more pPS cells in the culture). Thus, as usedherein, a cell culture begins with the initial seeding of one or moresuitable vessels with at least one pPS cell and ends when the lastsurviving progeny of the original founder(s) is harvested or dies.Seeding of one or more additional culture vessels with progeny of theoriginal founder cells is also considered to be a part of the originalcell culture.

Cytokine, as used herein, refers to a molecule secreted by a cell thataffects the behavior of another cell, or of the same cell, or both.

The term “embryoid bodies,” as used herein, refers to heterogeneousclusters comprising undifferentiated, differentiated and partlydifferentiated cells that appear when primate pluripotent stem cells areallowed to differentiate in a non-specific fashion in suspensioncultures or aggregates.

As used herein, “embryonic stem cell” (ES) refers to pluripotent stemcells that are derived from a blastocyst before substantialdifferentiation of the cells into the three germ layers. Except whereexplicitly required otherwise, the term includes primary tissue andestablished lines that bear phenotypic characteristics of ES cells, andprogeny of such lines that still have the capacity of producing progenycells bearing phenotypic traits of each of the three germ layers. The EScells may be human ES cells (hES). Prototype “human Embryonic Stemcells” (hES cells) are described by Thomson et al. (Science 282:1145,1998; U.S. Pat. No. 6,200,806) and include established cell linesdescribed therein.

Exogenous as used herein refers to agents added to a system, such as acell culture. The agent may be added to the system by the human hand.

As used herein, “feeder cells” refers to non-pPS cells that areco-cultured with pPS cells and provide support for the pPS cells.Support may include facilitating the growth and maintenance of the pPScell culture by providing the pPS cell culture with one or more cellfactors such that the pPS cells are maintained in a substantiallyundifferentiated state. Feeder cells may either have a different genomethan the pPS cells or the same genome as the pPS cells and may originatefrom a non-primate species, such as mouse, or may be of primate origin,e.g., human. Examples of feeder cells may include cells having thephenotype of connective tissue such as murine fibroblast cells, humanfibroblasts.

As used herein, “feeder-free” refers to a condition where the referencedcomposition contains no added feeder cells. To clarify, the termfeeder-free encompasses, inter alia, situations where primatepluripotent stem cells are passaged from a culture which may comprisesome feeders into a culture without added feeders even if some of thefeeders from the first culture are present in the second culture.

Hematopoietic lineage cells, as used herein, refers to cellsdifferentiated in vitro from pPS cells and/or their progeny and mayinclude one or more of the following: hemangioblasts, hematopoietic stemcells, common lymphoid progenitor cells, lymphocytes, common myeloidprogenitor cells (CMP), granulomonocytic progenitor cells (GMP),monocytes, macrophages, imDC and mDC.

Immunologically competent cell, as used herein, refers to a cell whichis capable of responding to an antigen. The responses may include forexample cell proliferation in response to antigen, secretion of one ormore cytokines in response to an antigen, expression of one or moretranscription factors in response to an antigen. Examples of animmunologically competent cell include lymphocytes.

In vitro progeny of a primate pluripotent stem cell, as used herein,refers to a cell that is differentiated in vitro from a pluripotentstate to a non-pluripotent state e.g. an immature DC, a mature DC.

As used herein, “primate pluripotent stem cells” (pPS) refers to cellsthat may be derived from any source and that are capable, underappropriate conditions, of producing primate progeny of different celltypes that are derivatives of all of the 3 germinal layers (endoderm,mesoderm, and ectoderm). pPS cells may have the ability to form ateratoma in 8-12 week old SCID mice and/or the ability to formidentifiable cells of all three germ layers in tissue culture. Includedin the definition of primate pluripotent stem cells are embryonic cellsof various types including human embryonic stem (hES) cells, (see, e.g.,Thomson et al. (1998) Science 282:1145) and human embryonic germ (hEG)cells (see, e.g., Shamblott et al., (1998) Proc. Natl. Acad. Sci. USA95:13726,); embryonic stem cells from other primates, such as Rhesusstem cells (see, e.g., Thomson et al., (1995) Proc. Natl. Acad. Sci. USA92:7844), marmoset stem cells (see, e.g., (1996) Thomson et al., Biol.Reprod. 55:254,), stem cells created by nuclear transfer technology(U.S. Patent Application Publication No. 2002/0046410), as well asinduced pluripotent stem cells (see, e.g. Yu et al., (2007) Science318:5858); Takahashi et al., (2007) Cell 131(5):861).

As used herein, “undifferentiated primate pluripotent stem cells” refersto a cell culture where a substantial proportion of primate pluripotentstem cells and their derivatives in the population display morphologicalcharacteristics of undifferentiated cells. It is understood thatcolonies of undifferentiated cells within the population may besurrounded by neighboring cells that are partly differentiated.

As used herein, “genetically altered”, “transfected”, or “geneticallytransformed” refer to a process where a polynucleotide has beentransferred into a cell by any suitable means of artificialmanipulation, or where the cell is a progeny of the originally alteredcell and has inherited the polynucleotide. The polynucleotide will oftencomprise a transcribable sequence encoding a protein of interest, whichenables the cell to express the protein at an elevated level or maycomprise a sequence encoding a molecule such as siRNA or antisense RNAthat affects the expression of a protein (either expressed by theunmodified cell or as the result of the introduction of anotherpolynucleotide sequence) without itself encoding a protein. The geneticalteration is said to be “inheritable” if progeny of the altered cellhave the same alteration.

Serum free, as used herein, refers tissue culture growth conditions thathave no added animal serum such fetal bovine serum, calf serum, horseserum, and no added commercially available serum replacement supplementssuch as B-27. Serum free includes, for example, media which may comprisehuman albumin, human transferrin and recombinant human insulin.

Spontaneous differentiated pPS cells, as used herein, refers to pPScells within a cell culture which randomly and spontaneouslydifferentiate to a non-pPS phenotype, i.e. express one or more markersnot expressed on pPS cells and/or fail to express one or more markersexpressed on a pPS cell.

Stromal cell, as used herein, refers to a cell which may be co-culturedwith another population, e.g. a pPS cell population in order tofacilitate the differentiation of the pPS cell population to a desiredphenotype, e.g. hematopoietic lineage cells by providing one or morecell factors. Stromal cells may be derived from the bone marrow of amammal. OP9 and S17 cells are examples of stromal cells.

Stromal cell free, as used herein, means that stromal cells, or mediaconditioned by stromal cells is not added to either the culture ofundifferentiated pPS cells or to a culture of pPS cells that aredifferentiating to hematopoietic lineage cells.

MHC-I and HLA-I are used interchangeably, as are MHC-II and HLA-II.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments the invention provides for improved methods forthe in vitro differentiation of pPS cells into hematopoietic lineagecells. Thus certain embodiments of the invention provide for definedconditions requiring a minimal number of exogenous factors (such ascytokines) suitable for differentiating pPS cells into hematopoieticlineage cells such as DC (including imDC and mDC). In some embodimentsthe invention provides for contacting the pPS cells with adifferentiation cocktail comprising a minimal number of exogenouscytokines, e.g. no more 7, no more 6, no more 5, no more 4, no more than3 exogenous cytokines thereby generating hematopoietic lineage cells. Inone embodiment the defined conditions may provide a differentiationcocktail comprising no more than four added exogenous cytokines, e.g.BMP-4, GM-CSF, SCF and VEGF. In other embodiments the defined conditionsmay provide for a differentiation cocktail comprising no more than threeadded exogenous cytokines, e.g., a) BMP-4, GM-CSF, SCF; b) BMP-4,GM-CSF, VEGF. In some embodiments a ligand to the respective cytokinereceptor may be substituted for the respective cytokine and/or providedin addition to the respective cytokine. In embodiments where thehematopoietic cells are imDC, the differentiation cocktail may furthercomprise IL-4. The imDC may be further contacted with a maturationcocktail to produce mDC.

In some embodiments the invention provides for simplified cultureconditions for differentiating pPS cells to hematopoietic lineage cellssuch as DC. Simplified culture conditions may include differentiatingpPS cells to DC in a tissue culture that is serum free, feeder free,stromal cell free and optionally does not require the addition ofexogenous IL-3. Differentiation of pPS to hematopoietic lineage cellsmay be performed by directly plating the pPS on a suitable solid surfacethereby avoiding the necessity of forming an embryo body (EB). Thesesimplified culture conditions eliminate the risk of exposure toinfectious agents and also provide a faster and less expensive method ofobtaining quantities of imDC cells that are sufficient for therapeuticand research applications.

Methods of Differentiating pPS Cells

Starting material for differentiating pPS cells into hematopoieticlineage cells include pPS cells which have been cultured serum free,feeder free and stromal cell free. Conditions for culturing pPS cellsfeeder free and serum free have been described see, e.g., Xu et al.,(2001) Nat Biotechnol 19:971; Li et al., (2005) Biotechnol Bioeng91:688. In some embodiments it may be advantageous to culture the pPScells under conditions that are suitable for the formation of cellularaggregates, e.g. embryoid bodies (EB). The formation of EBs has beenpreviously described see, e.g., U.S. Patent Publication No. 2006/0063255and PCT Publication No. WO 01/51616. Briefly undifferentiated pPS cellsmay be harvested by collagenase treatment, dissociated into clusters orstrips of cells, and passaged to non-adherent cell culture plates asaggregates. The harvested pPS cells may include some spontaneouslydifferentiated cells. It is contemplated that the number ofspontaneously differentiated cells may diminish over time as the cellsform EBs and then differentiate into hematopoietic lineage cells. Theaggregates may be fed with a suitable media, e.g. X-VIVO 10; X-VIVO 15.The pPS cells may be grown feeder free, serum free and stromal cell freeboth prior to and after formation of the EB.

In other embodiments the EB formation step may be skipped. Thus, pPScells may be directly plated onto a suitable support, such as a tissueculture flask or well, and cultured in a media comprising adifferentiation cocktail.

In various embodiments the invention provides methods of differentiatingpPS cells into cells of hematopoietic lineage and/or mesoderm cells. Thehematopoietic lineage cells may include imDC. In some embodiments theinvention provides for a differentiation cocktail comprising a pluralityof exogenously added cytokines suitable for differentiating pPS cells tohematopoietic lineage cells, e.g., BMP-4 and GM-CSF. Examples ofdifferentiation cocktails may include any of the following: a) BMP-4,GM-CSF, VEGF, SCF, FLT3L, TPO and IL-3; b) BMP-4, GM-CSF, VEGF, SCF andFLT3L; c) BMP-4, GM-CSF, VEGF, and SCF; d) BMP-4, GM-CSF, SCF; and e)BMP-4, GM-CSF, VEGF. In certain embodiments IL-4 may be used in additionto the cytokines recited above. In some embodiments ligands to one ormore cytokine receptors may be used in place of, or in addition to thecytokine.

It has also been discovered that various hematopoietic lineage cells maybe obtained by adjusting the amount of time the cells are exposed to thedifferentiation cocktail. In some embodiments of the invention pPS cellscultured for about 5 days with a differentiation cocktail in order todifferentiate the cells into a culture comprising mesoderm cells. Inanother embodiment of the invention pPS cells cultured for about 10 dayswith a differentiation cocktail in order to differentiate the cells intoa culture comprising hematopoietic stem cells. In still anotherembodiment of the invention pPS cells cultured for about 15 days with adifferentiation cocktail in order to differentiate the cells into aculture comprising a common myeloid progenitor cell. In yet anotherembodiment of the invention pPS cells cultured for about 20 days with adifferentiation cocktail in order to differentiate the cells into aculture comprising granulomonocytic progenitor cells. In still anotherembodiment of the invention pPS cells cultured for about 25 days with adifferentiation cocktail in order to differentiate the cells into aculture comprising monocytes. In a further embodiment of the inventionpPS cells cultured for about 30 days with a differentiation cocktail inorder to differentiate the cells into a culture comprising imDC.

Some embodiments of the invention provide for maturing imDC to mDC bycontacting the imDC with a suitable maturation cocktail comprising aplurality of exogenous cytokines. The maturation cocktail may compriseGM-CSF. Examples of suitable maturation cocktails include any of thefollowing: a) GM-CSF, TNFα, IL-1β, IFNγ, and PGE2; b) GM-CSF, TNFα,IL-1β, IFNγ, PGE2 and CD40L; c) GM-CSF, TNFα, IL-1β, IFNγ, PGE2, POLYI:C, and IFNα; d) GM-CSF, TNFα, IL-1β, IFNγ, POLY I:C, and IFNα; e)GM-CSF, TNFα, IL-1β, IFNγ, POLY I:C, IFNα, and CD40L; f) TNFα, IL-1β,PGE2 and IL-6; g) GM-CSF, IL-1β, PGE2, and, IFNγ; h) GM-CSF, TNFα, PGE2,and, IFNγ; i) GM-CSF, IL-1β, IFNγ and CD40L. In some embodiments ligandsto one or more cytokine receptors may be used in place of, and/or inaddition to the cytokine. Other methods, known in the art, may be usedto mature imDC to mDC. Examples include contacting imDC withlipopolysaccharide (LPS), contacting the imDC with CpG containingoligonucleotides, injecting the imDC into an area of inflammation withina subject.

The imDC may be cultured in the presence of the maturation cocktail, forat least about 12-15 hours, for at least about 1 day, for at least about2 days, for at least about 3 days to produce mDC. In some embodimentsthe imDC may be cultured in the presence of the maturation cocktail forabout 24 hours to produce mDC. In other embodiments the imDC may becultured in the presence of the maturation cocktail for about 48 hoursto produce mDC.

mDC may express one or more markers such as CD83, CD86, MHC I and MHCII, but not CD14 and may have functional properties similar to mature DCthat are differentiated in vivo. Functional properties may include thecapability to process and present antigen to an immunologicallycompetent cell. Processing and presenting antigen may include forexample the proteolysis of a target protein, as well as the expressionand processing of a nucleic acid encoding a target antigen. The mDC mayalso have the ability to migrate within peripheral and lymphoid tissue.Thus mDC differentiated from pPS cells according to the invention may beinduced to migrate in response to an appropriate stimulus such as MIP3β.The mDC may secrete one or more cytokines such as one or morepro-inflammatory cytokines. Exemplary cytokines secreted by DC accordingto the invention may include IL-12, IL-10 and IL-6.

Various embodiments of the invention described herein provide methods ofdifferentiating pPS cells into DC. It is contemplated that the methodsmay further comprise mitotically inactivating various types of cellsincluding unwanted pPS cells in a differentiated population as well ascells made according the methods described infra (e.g. any hematopoieticlineage cells, including mDC and imDC). Thus some embodiments of theinvention may comprise contacting the DC cells with a protein or peptideantigen or a nucleic acid encoding an antigen and contacting the DC e.g.an mDC, with a radiation source or a chemical agent suitable forinhibiting cell division. Exposure of the mDC to a radiation source orthe chemical agent may be desirable where the mDC are contained in apopulation of cells comprising at least one pPS cell. Irradiating thecells or treating the cells with the chemical agent will inhibit celldivision, while maintaining functionality of the mDC. Moreover, treatingthe cells with a radiation source or a chemical agent may minimize anyundesirable effects stemming from the presence of pPS cells in thepopulation.

In some embodiment the invention provides a method of differentiatingpPS cells into mesoderm comprising contacting the pPS cells with adifferentiation cocktail comprising a plurality of exogenous cytokinessuch as BMP-4, VEGF, SCF and optionally GM-CSF and culturing the cellsfor at least a day thereby differentiating pPS cells into mesoderm. Insome embodiments the cells may be cultured for at least about 2 days, atleast about 3 days, at least about 4 days, at least about 5 days withthe differentiation cocktail thereby differentiating the pPS cells intomesoderm. In certain embodiments the pPS cells may be cultured with adifferentiation cocktail for about 5 days in order to differentiate thepPS cells into mesoderm. In some embodiments the differentiationcocktail may optionally further comprise one or more of the following:FLT3L, TPO, IL-4 and IL-3. The mesoderm cells may express one or morefactors or markers expressed by mesoderm cells. For example increasedexpression of the mesoderm associated transcription factor, Brachyury,along with the decreased expression of pPS associated transcriptionfactor Oct4 and Tra-160 may be indicative of the differentiation of pPScells to mesoderm cells. Allowing the culture to continue to grow in thepresence of the differentiation cocktail may facilitate furtherdifferentiation of the mesoderm cells, e.g. into cells of hematopoieticlineage. Thus in some embodiments the cell culture may be grown in thepresence of the differentiation cocktail for a suitable length of timeto differentiate the cells beyond mesoderm cells and into otherhematopoietic lineage cells. For example the cells may be grown at leastabout 6 days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days with the differentiation cocktaildescribed herein thereby differentiating the pPS cells intohematopoietic stem cells. The cells may express one or more markersexpressed by hematopoietic stem cells. Suitable markers may includeCD45, CD34, and HoxB4. In yet further embodiments the cells may be grownat least about 22 days, at least about 23 days, at least about 24 days,at least about 25 days, at least about 26 days, at least about 27 days,at least about 28 days with the differentiation cocktail describedherein thereby differentiating the pPS cells into monocytes. The cellsmay express one or more markers expressed by monocytes. Suitable markersmay include CD14, CD45 and CD11c. In still further embodiments the cellsmay be grown at least about 20 days, at least about 23, at least about25 days, at least about 30 days, at least about 31 days, at least about32 days, at least about 33 days, with the differentiation cocktaildescribed herein thereby differentiating the pPS cells into imDC. Thecells may express one or more markers expressed by imDC. Suitablemarkers may include CD86, CD83, and MHC II.

In certain embodiments the invention provides a method ofdifferentiating pPS cells in hematopoietic lineage cells comprisingcontacting the pPS cells with one or more differentiation cocktails suchthat the pPS cells differentiate into one or more hematopoietic lineagecell types. The method may be comprised of multiple steps wherein one ormore of the steps results in the differentiation of intermediate celltypes of hematopoietic lineage. The invention contemplates not only theexecution of all of the steps set forth below, but also the execution ofone or more individual steps in order to attain a desired intermediateor precursor cell type of hematopoietic lineage.

In some embodiments the invention provides a method of differentiatingpPS cells into mesoderm comprising 1) contacting the pPS cells with afirst differentiation cocktail comprising BMP-4, VEGF, SCF andoptionally GM-CSF thereby differentiating pPS cells into mesoderm cells.The cells may be cultured with this differentiation cocktail for about1-5 days. In further embodiments the mesoderm cells from step 1) maythen be contacted with a second differentiation cocktail comprisingVEGF, SCF, GM-CSF thereby differentiating the mesoderm cells intohematopoietic stem cells. The cells may be cultured with thisdifferentiation cocktail for about 1-5 days. In further embodimentshematopoietic the stem cell may be further differentiated into a commonmyeloid progenitor (CMP) cell by contacting the hematopoietic stem cellwith a differentiation cocktail comprising GM-CSF. For this step thedifferentiation cocktail may further comprise SCF. The cells may becultured with this differentiation cocktail for about 1-10 days. In someembodiments the CMP may be further differentiated into a commongranulocytic/monocytic progenitor (GMP) cell by contacting the CMP witha third differentiation cocktail comprising GM-CSF. The cells may becultured with this differentiation cocktail for about 1-5 days. Infurther embodiments the GMP may be further differentiated into monocytesby contacting the GMP with a differentiation cocktail comprising GM-CSF.The cells may be cultured with this differentiation cocktail for about1-10 days. In still further embodiments the monocytes may be furtherdifferentiated into imDC by contacting the monocytes with adifferentiation cocktail comprising GM-CSF and IL-4. The cells may becultured with this differentiation cocktail for about 1-5 days. In yetfurther embodiments the imDC may be matured into mDC by contacting theimDC with any of the maturation cocktails described infra. The cells maybe cultured with the maturation cocktail from about 12-72 hours. In someembodiments the cells may be cultured with the maturation cocktail forabout 24 hours. In other embodiments the cells may be cultured with thematuration cocktail for about 48 hours.

In still other embodiments the invention provides a method ofdifferentiating pPS cells into imDC comprising contacting the pPS cellswith a differentiation cocktail comprising the following: 1) BMP-4ranging from about 10 ng/ml to about 75 ng/ml; and 2) GM-CSF rangingfrom about 25 ng/ml to about 75 ng/ml.

In still other embodiments the invention provides a method ofdifferentiating pPS cells into imDC comprising contacting the pPS cellswith a differentiation cocktail comprising the following: 1) BMP-4ranging from about 10 ng/ml to about 75 ng/ml; 2) VEGF ranging fromabout 25ng/ml to about 75 ng/ml; 3) SCF ranging from about 5 ng/ml toabout 50 ng/ml; and 4) GM-CSF ranging from about 25 ng/ml to about 75ng/ml.

In a further embodiment the invention provides a method of enriching amyeloid progenitor cell population comprising isolating a CD45+ Hipopulation from a cell culture comprising a CD45+ Hi cell population anda CD45+ low cell population. In a further embodiment the inventionprovides a method of isolating a granulocyte progenitor cell comprisingisolating a CD45+ low population from a cell culture comprising a CD45+Hi cell population and a CD45+ low cell population. High and low arerelative terms. Thus a CD45+ low cell population may refer to a cellshaving CD45 expression about 1-2 orders of magnitude above background,while the CD45+ Hi cells may refer to cells having CD45 expressiongreater than 2 orders of magnitude above background as measured usingany assay know in the art, e.g. immunofluorescence as measured using afluorescence detector, e.g. Fluorescent Activated Cell Sorter (FACS).Isolating the target cell population may be done using any means knownin the art. For example, the cell populations may be isolated using acommercially available (FACS). In some embodiments the cells may beisolated based on fluorescent intensity of a marker stained with alabeled ligand. The labeled ligand may attach directly to the cell orindirectly to the cell by virtue of another ligand attached to the cellby the human hand. The cell populations may be isolated based on sizeand density based on forward and side scatter on a cell sorter. As anexample CD45+ Hi and CD45+ low populations may be separated using a cellsorter based on size and granularity.

The cytokine combinations useful in carrying out various embodiments ofthe invention may be, used at any suitable final working concentrationto achieve the desired effect. For example, BMP-4 may be used at aconcentration ranging from about 1 ng/ml to about 120 ng/ml; from about5 ng/ml to about 100 ng/ml; from about 10 ng/ml to about 80 ng/ml; fromabout 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml.In some embodiments of the invention about 50 ng/ml of BMP-4 may beused. VEGF may be used at a concentration ranging from about 1 ng/ml toabout 120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 20ng/ml to about 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; fromabout 30 ng/ml to about 60 ng/ml. In some embodiments of the inventionabout 50 ng/ml of VEGF may be used. GM-CSF may be used at aconcentration ranging from about 1 ng/ml to about 120 ng/ml; from about5 ng/ml to about 100 ng/ml; from about 20 ng/ml to about 80 ng/ml; fromabout 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml.In some embodiments of the invention about 50 ng/ml of GM-CSF may beused. SCF may be used at a concentration ranging from about 1 ng/ml toabout 350 ng/ml; from about 5 ng/ml to about 300 ng/ml; from about 10ng/ml to about 250 ng/ml; from about 15 ng/ml to about 200 ng/ml; fromabout 20 ng/ml to about 150 ng/ml; from about 5 ng/ml to about 50 ng/ml.In some embodiments of the invention about 20 ng/ml of SCF may be used.FLT3L may be used at a concentration ranging from about 1 ng/ml to about350 ng/ml; from about 5 ng/ml to about 300 ng/ml; from about 10 ng/ml toabout 250 ng/ml; from about 15 ng/ml to about 200 ng/ml; from about 20ng/ml to about 150 ng/ml. In some embodiments of the invention about 20ng/ml of FLT3L may be used. IL-3 may be used at a concentration rangingfrom about 1 ng/ml to about 80 ng/ml; from about 5 ng/ml to about 75ng/ml; from about 10 ng/ml to about 50 ng/ml; from about 20 ng/ml toabout 40 ng/ml. In some embodiments of the invention about 25 ng/ml ofIL-3 may be used. TPO may be used at concentration ranging from about 1ng/ml to about 150 ng/ml; from about 5 ng/ml to about 100 ng/ml; fromabout 10 ng/ml to about 80 ng/ml; from about 20 ng/ml to about 60 ng/ml.In some embodiments of the invention about 20 ng/ml of TPO may be used.IL-4 may be used at a concentration ranging from about 1 ng/ml to about120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 20 ng/ml toabout 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; from about 30ng/ml to about 60 ng/ml. In some embodiments of the invention about 50ng/ml of IL-4 may be used.

In some embodiments of the invention a maturation cocktail comprising aplurality of cytokines may be used to mature imDC to mDC. Suitable finalworking concentrations of cytokine components of the maturation cocktailmay include any concentration which effectively matures imDC to mDC. Forexample IFNγ may be used at a concentration ranging from about 1 ng/mlto about 150 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 10ng/ml to about 100 ng/ml; from about 15 ng/ml to about 80 ng/ml; fromabout 20 ng/ml to about 60 ng/ml. In some embodiments of the inventionabout 25 ng/ml of IFNγ may be used. In other embodiments of theinvention about 10 ng/ml of IFNγ may be used. In other embodiments ofthe invention about 5 ng/ml of IFNγ may be used. TNFα may be used at aconcentration ranging from about 1 ng/ml to about 200 ng/ml; from about10 ng/ml to about 150 ng/ml; from about 20 ng/ml to about 100 ng/ml;from about 30 ng/ml to about 80 ng/ml; from about 40 ng/ml to about 75ng/ml. In some embodiments of the invention about 10 ng/ml of TNFα maybe used. IL-1β may be used at concentration ranging from about 1 ng/mlto about 200 ng/ml, from about 5 ng/ml to about 150 ng/ml; from about 8ng/ml to about 75 ng/ml; from about 10 ng/ml to about 50 ng/m. In someembodiments of the invention about 10 ng/ml of IL-1β may be used. PGE2may be used at a concentration ranging from about 0.1 ug/ml to about 150ug/ml; from about 0.5 ug/ml to about 100 ug/ml; from about 0.8 ug/ml toabout 75 ug/ml; from about 1 ug/ml to about 50 ug/ml. In someembodiments of the invention about 1 ug/ml of PGE2 may be used. Poly I:Cmay be used a concentration ranging from about 1 ug/ml to about 50ug/ml, from about 5 ug/ml to about 40 ug/ml from about 10 ug/ml to about30 ug/ml, form about 15 ug/ml to about 25 ug/ml. In some embodiments ofthe invention about 20 ug/ml of Poly I:C may be used.

In certain embodiments the invention provides for the differentiation ofpPS cells in hematopoietic lineage cells wherein at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99% of cells express one or more markers or factors that are expressedby cells of hematopoietic lineage.

Cell Cultures Comprising Primate Pluripotent Stem Cells and TheirDifferentiated Progeny

In certain embodiments the invention provides a cell culture comprisinga first population of cells comprising pPS cells and a second populationof cells comprising hematopoietic lineage cells and/or mesoderm cells.The hematopoietic lineage cells and/or mesoderm cells may arise in theculture as a result of specific growth conditions which favor thedifferentiation of pPS cells into a target cell type, e.g. mesodermcells, myeloid precursor cells, monocytes, dendritic cells and the like.The growth conditions may include providing one or more differentiationcocktails (as described infra) and in some embodiments a maturationcocktail (as described infra). The cell culture may be free of one ormore of the following: feeder cells, stromal cells, animal serum and/orcommercially available serum replacements such as B27, and exogenousIL-3.

In some embodiments the second population of cells may comprise mesodermcells. In the developing embryo mesoderm is positioned between theectoderm and endoderm. Connective tissue, bone, cartilage, muscle,hematopoietic lineage cells, blood and blood vessels, lymphatics,lymphoid organs, notochord, pleura, pericardium, peritoneum, kidneys andgonads all originate from the mesoderm. Mesoderm cells may expressvarious markers including expression of the transcription factorbrachyury. Expression levels of brachyury may increase about three tosix fold when compared to pPS cells prior to differentiation to mesodermcells. Other markers for mesoderm may include goosecoid. Goosecoid amember of the bicoid subfamily of the paired (PRD) homeobox family ofproteins. The encoded protein acts as a transcription factor and may beautoregulatory.

In other embodiments the hematopoietic lineage cells may comprisehemangioblast cells. Hemangioblast cells have the ability todifferentiate further into lymphoid cells of various types, myeloidcells of various types, as well as endothelial cells. Hemangioblasts mayexpress CD34 and CD133. Loges et al., (2004). Stem Cells and Development13 (1): 229. Other markers for hemangioblasts include Flk-1 which is akinase insert domain receptor.

In yet other embodiments the hematopoietic lineage cells may comprisehematopoietic stern cells. Hematopoietic stern cells may be able todifferentiate into any cell type found in the blood, including lymphoidcells, (whose progeny includes T cells and B lymphocytes) and myeloidcells (whose progeny includes granulocytes of various types, monocytes,macrophages, DC, megakaryocytes, platelets, erythroblasts, anderythrocytes). Markers for hematopoietic stem cells may include CD34+,CD59+, Thy1/CD90+, CD38^(lo/−), C-kit/CD117^(−/lo). In certainembodiments of the invention the percentage of cells expressing at leastone marker associated with hematopoietic stem cells ranges from about 1%to about 20%, from about 5% to about 17%, from about 10% to about 15%.In some embodiments of the invention about 15% of the cells in the cellculture express at least one marker associated with hematopoietic stemcells.

In further embodiments the hematopoietic lineage cells may comprisecommon myeloid progenitor cells. Myeloid progenitor cells may, underappropriate growth conditions, differentiate into various myeloid cellsincluding granulocytes, monocytes, macrophages, DC,megakaryocyte/erythrocyte progenitor cells. Markers for myeloidprogenitor cells may include CD13, CD34, IL-3Rα (CD123), and CD45RA. Incertain embodiments of the invention the percentage of cells expressingat least one marker associated with myeloid progenitor cells ranges fromabout 1% to about 50%, from about 5% to about 45%, from about 6% toabout 38%. In some embodiments of the invention about 35% of the cellsin the cell culture express at least one marker associated with myeloidprogenitor cells.

In still other embodiments the hematopoietic lineage cells may comprisegranulomonocytic progenitor cells. Granulomonocytic progenitor cellsmay, under appropriate conditions differentiate into granulocytes,monocytes, macrophages and DC. Markers for granulomonocytic progenitorcells may include CD64 (EP0708336).

In further embodiments the hematopoietic lineage cells may comprisemonocytes. Under appropriate growth conditions monocytes maydifferentiate into DC, macrophages and granulocytes cells. Markers formonocytes may include CD14, CD45^(hi), CD11a, CD11b, and CD15. Themonocyte morphology may include the presence of a large bi-lobednucleus. In certain embodiments of the invention the percentage of cellsexpressing at least one marker associated with monocytes ranges fromabout 1% to about 75%, from about 5% to about 70%, from about 10% toabout 65%. In some embodiments of the invention about 65% of the cellsin the cell culture express at least one marker associated withmonocytes.

In still further embodiments the hematopoietic lineage cells maycomprise imDC. imDC have the ability to take up and process antigen.Under appropriate growth conditions imDC may undergo maturation tobecome mDC suitable for presenting antigens to an immunologicallycompetent cell. Markers for imDC may include CD11c^(hi), CD11b, MHC I,MHC II^(lo), CD14^(−/lo), CD205⁻, and CD83^(lo). In certain embodimentsof the invention the percentage of cells expressing at least one markerassociated with imDC ranges from about 10% to about 99%, from about 20%to about 99%. In certain embodiments of the invention at least about90%, about 80% about 70%, about 60%, about 50% about 40% about 30%,about 20% about 10% of the cells in the cell culture express at leastone marker associated with imDC.

In yet other embodiments the hematopoietic lineage cells may comprisemDC. mDC may have the ability to migrate in response to an appropriatestimuli e.g, MIP3β and to present antigen to an immunologicallycompetent cell such as a T lymphocyte. mDC may have a distinctivemorphology that include the presence of branched projections ordendrites which emanate out from the cell. Markers for mDC may includeCD83, CCR7, CD11c^(hi), CD205, CD86, CD40, MHC I, MFIC II and CD14⁻. Incertain embodiments of the invention the percentage of cells expressingat least one marker associated with mDC ranges from about 10% to about99%, from about 20% to about 99%. In certain embodiments of theinvention at least about 90%, about 80%, about 70%, about 60%, about50%, about 40%, about 30%, about 20%, about 10%, of the cells in thecell culture express at least one marker associated with mDC.

Tissue-specific markers may be detected using suitable immunologicaltechniques—such as flow immunocytometry or affinity adsorption forcell-surface markers, immunocytochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. Expression of anantigen by a cell is said to be antibody-detectable if a significantlydetectable amount of antibody will bind to the antigen in a standardimmunocytochemistry or flow cytometry assay, optionally after fixationof the cells, and optionally using a labeled secondary antibody.

The expression of tissue-specific gene products may also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods using publicly available sequence data (GenBank).Expression of tissue-specific markers as detected at the protein or mRNAlevel is considered positive if the level is at least about 2-fold, morethan about 10- or about 50-fold above that of an undifferentiatedprimate pluripotent stem cell.

The expression of tissue-specific gene products may also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for the particular markers listed in this disclosure canbe obtained from public databases such as GenBank. Expression at themRNA level is said to be “detectable” according to one of the assaysdescribed in this disclosure if the performance of the assay on cellsamples according to standard procedures in a typical controlledexperiment results in clearly discernable hybridization or amplificationproduct. Expression of tissue-specific markers as detected at theprotein or mRNA level is considered positive if the level is at leastabout 2-fold, more than about 10- or about 50-fold above that of anundifferentiated primate pluripotent stem cell.

Once markers have been identified on the surface of cells of the desiredphenotype, they can be used for immunoselection to further enrich thepopulation by techniques such as immunopanning or antibody-mediatedFACS.

Irradiation of DC Differentiated from pPS Cells

The invention contemplates methods of irradiating populations of cellscomprising mDC or imDC differentiated in vitro from pPS cells, i.e. arethe in vitro progeny of pPS cells, as well as irradiated cell culturescomprising mDC or imDC differentiated in vitro from pPS cells. Otherembodiments of the invention contemplate irradiated immuno-modulatorypreparations and methods of making the same as well as methods ofstimulating an immune response using an irradiated cell populationcomprising mDC. Still other embodiments of the invention contemplatekits comprising irradiated mDC.

Irradiating mDC differentiated from pPS cells inhibits any further celldivision thereby diminishing any risk, such as teratomas formation,posed by cells (e.g. pPS cells) which have not fully differentiated intomDC. The irradiated mDC may maintain functional properties associatedwith non-irradiated DC such as PBMC derived DC, and thus irradiated mDCmay be capable of processing and presenting antigen to animmunologically competent cell and causing that cell to respond to thepresented antigen. The irradiated mDC may also maintain the ability tomigrate in response to chemotactic stimuli. Furthermore, the irradiatedmDC may also continue to express markers typically found on mDC, e.g.,PBMC derived mDC. These markers may include HLA-II, HLA-I, and CD83. Itis further contemplated that mDC according to the invention may becontacted with an antigen or a nucleic acid encoding an antigen prior toexposure to a radiation source.

In some embodiments an mDC may be contacted with antigen, e.g. a proteinor a peptide and then irradiated. In other embodiments an mDC may becontacted, e.g. electroporated or contacted using any other suitabletransfection means, with a nucleic acid such as an RNA molecule and thenirradiated. In some embodiments the cells may be contacted with thenucleic acid and then permitted to rest for about 24 hours (e.g. at 37°C. and 5% CO₂). The cells may then be placed in a suitable cryo media(e.g. one comprising DMSO) and then frozen at about −80° C. The frozencells may then be irradiated (e.g. on dry ice) and then stored frozen(e.g. in liquid nitrogen) until further use is required.

Any suitable source of radiation may be used to irradiate mDC accordingto the invention. In one embodiment the radiation source may be anionizing radiation source. As an example an X-ray may provide a suitablesource of radiation. Other types of radiation which may be suitableinclude UV irradiation e.g. gamma irradiation.

The cell population comprising mDC differentiated in vitro from pPScells may be irradiated for a suitable length of time e.g. such thatcell division is inhibited. Parameters such as radiation dosage, cellpopulation size and time of exposure may be optimized empirically andthen tested by culturing cells post radiation and determining whether ornot the cells continue to divide. Determination of cell division may beaccomplished by counting cells manually using a hemacytometer.Alternatively an automated cell counter may be used.

When the radiation source is an X-ray a suitable radiation dose mayrange from about 300 rad to about 3500 rad; from about 400 rad to about3000 rad; from about 500 rad to about 2500 rad; from about 500 rad toabout 2000 rad; from about 400 rad to about 1500 rad. In one embodimentabout 2000 rad are applied to a population of cells comprising mDC. Inanother embodiment about 1500 rad are applied to a population of cellscomprising mDC. In a further embodiment about 1000 rad are applied to apopulation of cells comprising mDC. In still another embodiment about500 rad are applied to a population of cells comprising mDC.

Where the radiation source is UV irradiation a suitable dose may rangefrom about 10 J/m² to about 3,000 J/m²; from about 20 J/m² to about2,000 J/m²; from about 25 J/m² to about 1,500 J/m²; from about 30 J/m²to about 500 J/m²; from about 50 J/m² to about 200 J/m². In someembodiments about 50 J/m² may be used. In other embodiments about 100J/m² may be used. In other embodiments about 200 J/m² may be used. Instill other embodiments about 300 J/m² may be used. In yet otherembodiments about 500 J/m² may be used.

Where the radiation source is an X-ray, the cells may be suspended in asuitable media or buffer prior to exposure to the radiation source. Asuitable media would include any commercially available media forgrowing or differentiating stem cells. As an example AIM V media(Invitrogen, Carlsbad, Calif.) may be used. A suitable buffer mayinclude any isotonic buffer, e.g. PBS. The volume of media used willdepend on the size of the cell population to be irradiated. For apopulation of cells ranging from about 3.0×10⁵ to about 4.0×10⁵ asuitable volume may range from about 5-20 mls of media or buffer. In oneembodiment about 15 mls of media or buffer may be used.

Where the radiation source is a UV light the cells may grown attached toa substrate such as a tissue culture flask and exposed to the radiationsource. The cells may be maintained in a suitable buffer or media duringexposure to the radiation.

In one embodiment a population of cells ranging from about 3.0×10⁵ cellsto about 4.0×10⁵ cells is irradiated with about 2000 rad from an X-raysource. In another embodiment a population of cells ranging from about3.0×10⁵ cells to about 4.0×10⁵ cells is irradiated with about 1500 radfrom an X-ray source. In yet another embodiment a population of cellsranging from about 3.0×10⁵ cells to about 4.0×5 cells is irradiated withabout 1000 rad from an X-ray source. In still another embodiment apopulation of cells ranging from about 3.0×10⁵ cells to about 4.0×10⁵cells is irradiated with about 500 rad from an X-ray source. The cellsmay be comprised of mDC differentiated in vitro from pPS cells.

It is also contemplated that a chemical agent suitable for inhibitingcell division may be substituted for the radiation source. Thus cellpopulations comprising mDC differentiated in vitro from pPS cells may becontacted with a chemical agent suitable for inhibiting cell division.Examples of suitable chemicals include chemotherapeutics such asmitomycin C and cisplatin. Other suitable chemicals may include one ormore of the following: arabinoside, fluoro-deoxyuridine and uridine.

Systems for Producing Dendritic Cells

In certain embodiments the invention contemplates a system for the invitro production of mature dendritic cells comprising a) a firstisolated cell population comprising pPS cells and b) a second isolatedcell population comprising mature dendritic cells which are the in vitroprogeny of a portion of the first population of cells.

In other embodiments the invention contemplates a system for theproduction of mitotically inactive antigen presenting cells comprisinga) a first isolated cell population comprising pPS cells and b) a secondisolated cell population comprising mitotically inactivated maturedendritic cells which are the in vitro progeny of a portion of the firstisolated cell population.

The mDC may be differentiated in vitro from a portion of the pPS cellsand a portion of the first isolated cell population comprising the pPScells can be held in reserve, used to make more of the second isolatedpopulation by differentiating the first population of cells in vitro.The system contemplates that the first population of cells comprisingpPS cells may include a portion of the population that may bedifferentiated into DC using the methods described herein, and a portionof the population that may be reserved for future use e.g. maintained inculture in an undifferentiated state or frozen (in a suitable media) inaliquots and stored at −80° C. or in liquid nitrogen. Because the pPScells are capable of replicating in culture in an undifferentiated(pluripotent) state indefinitely, the system provides a means ofproducing a limitless supply of differentiated DC according to themethods described herein. Moreover, because the DC, e.g. imDCdifferentiated in vitro from the pPS cells may also replicate in culturethe system provides a second population which is capable of producingadditional DC. The system thus provides a means of continually producinglarge quantities of a uniform product such as a DC. The system may alsoinclude embodiments in which one or both populations are mitoticallyinactivated as described infra.

Characteristic markers and morphology of both the first population ofcells comprising pPS cells and the second population of cells comprisingDC cells are described herein. The DC cells may be mDC loaded with anantigen of interest, e.g. a tumor antigen such as hTERT. The DC cellsmay be irradiated according to the invention in order to mitoticallyinactivate the cells and thus diminish any potential risk of pPS cellswhich may be present in the second population of cells comprising theDC. In certain embodiments at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95% of the mDC may express one or moremarkers chosen from CD83, CD86, MHC II and CCR7.

Kits

In certain embodiments the invention provides a kit for stimulating animmune response. In one embodiment the kit may comprise a cell culturecomprising pPS cells and DC, and one or more containers. Optionally thekit may comprise one or more of the following: a) instructions forstimulating an immune response; b) instructions for culturing the DC; c)a maturation cocktail, where the provided DC are imDC; c) one or moresuitable culture vessels; d) one more antigens for stimulating an immuneresponse; e) one or more immunologically competent cells; and f)suitable reagents for measuring the stimulated immune response. The kitmay be used to stimulate an immune response in vitro or in vivo. The DCmay be imDC or mDC. In some embodiments the DC may be provided frozen.The cells may be frozen in liquid nitrogen and stored at about −140° C.Alternatively, the DC may be packaged and stored under refrigeration,e.g. at about 4° C. The maturation cocktail may be supplied premixed orwith each of its components packaged separately. The antigen may beprovided as a protein or peptide or as nucleic acid, e.g., DNA, RNAencoding the antigen. The kit may also provide instructions for loadingthe DC with the antigen. Loading refers to contacting the DC with theantigen such that it is presented to an immunologically competent cell.The kit may further comprise instructions for growing the DC in culture,for contacting the DC with antigen, for contacting the DC withimmunologically competent cells. Suitable reagents for detecting astimulated immune response may include ³H thymidine for measuring cellproliferation, antibodies to cytokines secreted during an immuneresponse to an antigen such as IL-2, IFN.

In another embodiment the kit may comprise an irradiated mDCdifferentiated in vitro from a pPS cell and one or more containers.Optionally the kit may comprise one or more of the following: a)instructions for stimulating an immune response; b) one more preloadedantigens for stimulating an immune response; c) one or moreimmunologically competent cells; and d) suitable reagents for measuringthe stimulated immune response. The kit may be used to stimulate animmune response in vitro or in vivo. The preloaded antigen may beprovided as a protein or peptide or as nucleic acid, e.g., DNA, RNAencoding the antigen. Loading refers to contacting the DC with theantigen such that it is presented to an immunologically competent cell.Suitable reagents for detecting a stimulated immune response may include³H thymidine for measuring cell proliferation, antibodies to cytokinessecreted during an immune response to an antigen such as IL-2, IFN. Inother embodiments the invention provides a kit, as described above wherechemically treated mDC may be substituted for the irradiated mDC. Thechemically treated mDC may be mDC that have been contacted with achemical agent suitable for inhibiting cell division. Suitable chemicalagents may include mitomycins such as mitomycin C.

Uses of ES-Differentiated Hematopoietic-Lineage Cells

This invention provides a method to produce large numbers of cells ofthe hematopoietic and/or mesoderm lineage. These cell populations can beused for a number of important research, development, and commercialpurposes.

Screening

The cells of this invention can be used commercially to screen forfactors (such as solvents, small molecule drugs, peptides,oligonucleotides) or environmental conditions (such as cultureconditions or manipulation) that affect the characteristics of suchcells and their various progeny. Characteristics may include phenotypicor functional traits of the cells.

In some applications, pPS cells (undifferentiated or differentiated) areused to screen factors that promote maturation into later-stagehematopoietic precursors, or terminally differentiated cells, or topromote proliferation and maintenance of such cells in long-termculture. For example, candidate maturation factors or growth factors aretested by adding them to cells in different wells, and then determiningany phenotypic change that results, according to desirable criteria forfurther culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on hematopoietic lineage cellsand/or mesoderm cells. Screening may be done either because the compoundis designed to have a pharmacological effect on the cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on cells of this tissue type. Other screening applications couldinclude screening compounds for carcinogenic or other toxic effects. Thescreening can be conducted using any of the precursor cells orterminally differentiated cells of the invention in order to determineif the target compound has a beneficial or harmful effect on the targetcell.

The reader is referred generally to the standard textbook In vitroMethods in Pharmaceutical Research, Academic Press, 1997. Assessment ofthe activity of candidate pharmaceutical compounds generally involvescombining the cells of this invention with the candidate compound,either alone or in combination with other drugs. The investigatordetermines any change in the morphology, marker phenotype, or functionalactivity of the cells that is attributable to the compound (comparedwith untreated cells or cells treated with an inert compound), and thencorrelates the effect of the compound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in In vitro Methods in PharmaceuticalResearch, Academic Press, 1997) for further elaboration.

Where an effect is observed, the concentration of the compound can betitrated to determine the median effective dose (ED₅₀).

Modulation of an Immune Response

In certain embodiments the invention provides a method of stimulating animmune response to an antigen comprising contacting a cell according tothe invention, e.g., a DC differentiated from pPS cells, with anantigen. The antigen may be comprised of a protein or peptide oralternatively it may be comprised of a nucleic acid e.g. DNA, RNA. Wherethe antigen is a protein or peptide the dendritic cell will take up theprotein or peptide and process it for presentation in the context of theMHC. Typically processing includes proteolysis so that the antigen willfit in the MHC groove. Where the antigen is a protein the DC cell may bean imDC. Where the antigen is a peptide fragment of a full lengthprotein the DC may be a mDC. Where the antigen is a nucleic acid theinvention contemplates using any means known in the art for transportingthe nucleic acid across the cell membrane for delivery into thecytoplasm. In one embodiment the cells may be electroporated to allowthe nucleic acid to cross the cell membrane. In some embodiments whereelectroporation is used to contact the cell with an antigen a suitablecell may be an imDC. In other embodiments where electroporation is usedto contact the cell with an antigen a suitable cell may be an mDC. Thecells may be electroporated using Gene Pulse Xcell (Bio-RadLaboratories, Hercules, Calif.) with the following parameters: 300V, 150uF, and 100 Ohms. Protein expression levels may be determined by flowcytometry or western blot methods. Where the electroporated cell is animDC the cells may be contacted with a maturation cocktail as describedherein such that the imDC mature into mDC.

In another embodiment a viral vector may be used to transport thenucleic acid encoding the antigen into the cell, e.g., a mDC, an imDC.Where a viral vector is used to contact the cell with an antigen asuitable cell may be imDC. Examples of suitable viral vectors includeadenoviral vectors and pox viral vectors. In other embodimentscommercially available transfection reagents may be used to transportthe nucleic acid encoding the antigen into the cell. Suitable examplesinclude cationic lipid formulations such as Lipofectamine®.

The invention contemplates using antigens from any source. Thus theantigen may be a tumor antigen such as human teleomerase reversetranscriptase (hTERT) or an antigen expressed by infectious agent suchas a virus, a bacterium, or a parasite.

The mDC may then be contacted, either in vivo or in vitro, with animmunologically competent cell such as a lymphocyte. The immune responseof the lymphocyte may be monitored by measuring cell proliferation ofthe immunologically competent cell (e.g., by ³H thymidine incorporation)and/or cytokine production (e.g. IL-2, IFN, IL-6, IL-12) by either themDC or the immunologically competent cell. These studies may be usefulin tailoring the type and extent of the immune response to the antigen.These studies may also be useful in selecting the best epitope of theantigen for eliciting the most appropriate immune response. The immuneresponse may be stimulated in vitro or in vivo using an appropriateanimal model.

To determine the suitability of cell compositions for therapeuticadministration, the cells can first be tested in a suitable animalmodel. Suitable animal models may include a mouse with a humanizedimmune system. See, e.g., Goldstein (2008) AIDS Res Ther 5(1):3. mDCprimed with a specific antigen may be administered to an animal todetermine whether or not the animal is able to mount a specific immuneresponse to the antigen. The animal and the DC may be matched orpartially matched at the MHC I locus. Dosing, administration andformulation of the antigen and of the cells may be studied to tailor theimmune response to the antigen and migration of the administered cellswithin the lymphatic system may be monitored. The extent of the immuneresponse may be characterized in terms of cytokine production as well aslymphocyte proliferation in response to the antigen. The animal may bemonitored for an antibody response against the antigen as well as forany atypical immune reaction, e.g. hypersensitivity, autoimmunereaction. The antibody generated may be isolated for use as a researchreagent or therapeutic agent.

imDC are known to induce antigen specific tolerance, see, e.g., Cools etal., (2007) J Leukoc Biol 82(6):1365. Thus imDC, as described herein,may be used to induce tolerance within a subject. The imDC cells may becontacted with antigen, e.g., a protein or peptide antigen or a nucleicacid encoding an antigen as described above. The cells may then beadministered to a subject to induce tolerance in the subject.Alternatively, the imDC may be matured into mDC and used to stimulate animmune response.

Reconstitution of Hematopoietic Cells

Hematopoietic lineage cells made according to the invention may be usedto reconstitute one or more hematopoietic cells populations in asubject. As an example myeloid progenitor cells may be used toameliorate one or more symptoms associated with cytopenia byreconstituting a cell population that is deficient. For example myeloidprogenitors may be used improve the condition of a subject with lowplatelet count, or low erythrocyte count. As another examplehematopoietic lineage cells, such as myeloid precursor cells may be usedto improve one or more symptoms of a subject with a genetic defect, suchas a defect relating to a clotting factor. Thus cells made according tosome embodiments of the invention may be used increase the level of aclotting factor such as factor VIII or factor IX. In other embodimentsthe hematopoietic lineage cells may be used to reconstitute a lymphocytepopulation, e.g. a CD4 lymphocyte population in a patient with HIV.

Administration to Humans

The mDC produced according to the invention are functionally comparableto mDC isolated from PBMCs. For example the mDC of the invention cantake up process and present antigen; stimulate T cell proliferation inresponse to presentation of a specific antigen and can induce antigenspecific T cell mediated cytolysis of target cells. Additionally,functionality of the mDC according to the invention is maintained evenafter irradiation. The mDC of the invention may thus provide a source ofDC for administration to a human subject in order to stimulate an immuneresponse to a specific antigen while minimizing the risk of exposure toundifferentiated cells and pathogenic agents.

mDC according to this invention may be administered to a human subjectto stimulate an immune response in the subject. Prior to administration,the imDC may be contacted with an antigen of interest and then maturedinto mDC. The antigen may be internalized and processed such that it ispresented on the cell surface in the context of MHC I and/or MHC II andthus may stimulate a specific immune response to the antigen. In someembodiments the specific immune response may have a therapeutic effect.In other embodiments the immune response may provide a prophylacticeffect. In still other embodiments the specific immune response mayprovide a source of antigen specific cells such as cytotoxic T cells, orB lymphocytes or antibodies which specifically recognize the antigen.Administration of the cells according to the invention may be byintravenous, intradermal or intramuscular injection. In otherembodiments the cells may be administered subcutaneously. The cells maybe formulated with an appropriate buffer, such as PBS and/or anappropriate excipient. The cells may be formulated with a suitableadjuvant. Examples of suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences by E. W. Martin 20th Edition.Baltimore, Md.: Lippincott Williams & Wilkins, 2000.

For general principles in medicinal formulation, the reader is referredto Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000. Choice of the cellular excipientand any accompanying elements of the composition will be adapted inaccordance with the route and device used for administration.

Pharmaceutical compositions of this invention may optionally be packagedin a suitable container with written instructions for a desired purpose,such as the reconstitution of hematopoietic-lineage cell function toimprove a disease condition or to stimulate an immune response.

Other Uses

The cells of this invention may be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. For example, hematopoietic-lineage cells arecollected by centrifugation at 1000 rpm for 5 minutes, and then mRNA isprepared and reverse transcribed. Expression patterns of thehematopoietic-lineage cells may be compared with other cell types, e.g.,pPS cells, by microarray analysis, reviewed generally by Fritz et al.,(2000) Science 288:316. Because the cells are virtually geneticallyidentical to the parental pPS cell line from which they differentiatedthey provide a particularly well suited system for studying genesinvolved in the differentiation and maturation of hematopoietic lineagecells. For example nucleic acid libraries from the parental cell lineand the hematopoietic progeny may be prepared and subtractivehybridization may be employed to isolate genes important indifferentiation and maturation of the progeny cells.

The differentiated cells of this invention can also be used to prepareantibodies that are specific for markers of hematopoietic-lineage cells.Polyclonal antibodies can be prepared by injecting a vertebrate animalwith cells of this invention in an immunogenic form. Production ofmonoclonal antibodies is described in such standard references as Harlow& Lane (1988) Antibodies: A Laboratory Manual, U.S. Pat. Nos. 4,491,632,4,472,500 and 4,444,887, and Methods in Enzymology 73B:3 (1981).

Primate Pluripotent Stem Cells

The present invention provides methods for differentiating pPS cellsinto hematopoietic-lineage cells. pPS cells include any primatepluripotent cell. A pluripotent cell will, under appropriate growthconditions, be able to form at least one cell type from each of thethree primary germ layers: mesoderm, endoderm and ectoderm. The pPScells may originate from pre-embryonic, embryonic or fetal tissue ormature differentiated cells. Alternatively, an established pPS cell linemay be a suitable source of cells for practicing the invention.Typically, the pPS cells are not derived from a malignant source. pPScells will form teratomas when implanted in an immuno-deficient mouse,e.g. a SCID mouse.

Under the microscope, primate pluripotent stem cells appear with highnuclear/cytoplasmic ratios, prominent nucleoli, and compact colonyformation with poorly discernable cell junctions. Primate pluripotentstem cells typically express the stage-specific embryonic antigens(SSEA) 3 and 4, and markers detectable using antibodies designatedTra-1-60 and Tra-1-81. Undifferentiated human embryonic stem cells alsotypically express the transcription factor Oct-3/4, Cripto,gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein(PODXL), nanog and telomerase reverse transcriptase, e.g., hTERT (US2003/0224411 A1), as detected by RT-PCR.

pPS cells that may be used in any of the embodiments of the inventioninclude, but are not limited to, embryonic stem cells such as humanembryonic stem cells (hES). Embryonic stem cells can be isolated fromblastocysts of a primate species (U.S. Pat. No. 5,843,780; Thomson etal., (1995) Proc. Natl. Acad. Sci. USA 92:7844,). hES cells can beprepared from human blastocyst cells using, for example, the techniquesdescribed in U.S. Pat. No. 6,200,806; Thomson et al., (1998) Science282:1145; Thomson et al. (1998) Curr. Top. Dev. Biol. 38:133 ff. andReubinoff et al., (2000) Nature Biotech. 18:399.

Other primate pluripotent stem cell types include, but are not limitedto, primitive ectoderm-like (EPL) cells, described in WO 01/51610 andhuman embryonic germ (hEG) cells (Shamblott et al., (1998) Proc. Natl.Acad. Sci. USA 95:13726).

pPS cells suitable for use in any of the embodiments of the inventionalso include induced primate pluripotent stem (iPS) cells. iPS cellsrefer to cells that are genetically modified, e.g., by transfection withone or more appropriate vectors, such that they attain the phenotype ofa pPS cell. Phenotypic traits attained by these reprogrammed cellsinclude morphology resembling stem cells isolated from a blastocyst,surface antigen expression, gene expression and telomerase activity thatare all similar blastocyst derived cells. The iPS cells may have theability to differentiate into at least one cell type from each of theprimary germ layers: ectoderm, endoderm and mesoderm. The iPS cells mayalso form teratomas when injected into immuno-deficient mice, e.g., SCIDmice. (Takahashi et al., (2007) Cell 131(5):861; Yu et al., (2007)Science 318:1917).

Embryonic stem cells used in the invention may be chosen fromestablished embryonic stem cell lines or may be obtained directly fromprimary embryonic tissue. A large number of embryonic stem cell lineshave been established including, but not limited to, H1, H7, H9, H13 orH14 (reference Thompson); hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen,Inc., Athens, Ga.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (from ESCell International, Inc., Singapore); HSF-1, HSF-6 (from University ofCalifornia at San Francisco); I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J3.2 (derived at the Technion-Israel Institute of Technology, Haifa,Israel); UCSF-1 and UCSF-2 (Genbacev et al., (2005) Fertil. Steril.83(5):1517); lines HUES 1-17 (Cowan et al., (2004) NEJM 350(13):1353);and line ACT-14 (Klimanskaya et al., (2005) Lancet, 365(9471):1636).

In certain embodiments, pPS cells used in the present invention may havebeen derived in a feeder-free manner (see, e.g., Klimanskaya et al.,(2005) Lancet, 365(9471):1636). In certain embodiments the pPS may becultured prior to use in a serum free environment.

Culture Conditions for Primate Pluripotent Stem Cells

pPS cells may be cultured using a variety of substrates, media, andother supplements and factors known in the art. In some embodiments asuitable substrate may include a matrix comprised of one or more of thefollowing: laminin, collagen, fibronectin, vitronectin, heparin sulfateproteoglycan. In some embodiments the matrix may comprise a solubleextract of the basement membrane from a murine EHS sarcoma which iscommercially available as Matrigel™ (BD Biosciences, San Jose, Calif.).In other embodiments the matrix may comprise one more isolated matrixproteins of human, humanized, or murine origin, e.g. CELLstart™(Invitrogen, Carlsbad, Calif.). Primate pluripotent stem cells can bepropagated continuously in culture, using culture conditions thatpromote proliferation while inhibiting differentiation. Exemplary mediummay be made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of eitherdefined fetal bovine serum (FBS, Hyclone) or serum replacement (US2002/0076747 A1, Life Technologies Inc.), 1% non-essential amino acids,1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Other suitable mediainclude serum free defined media such as X-VIVO™ 10 (Lonza,Walkersville, Md.).

In certain embodiments, pPS cells may be maintained in anundifferentiated state without added feeder cells (see, e.g., (2004)Rosier et al., Dev. Dynam. 229:259). Feeder-free cultures are typicallysupported by a nutrient medium containing factors that promoteproliferation of the cells without differentiation (see, e.g., U.S. Pat.No. 6,800,480). In certain embodiments, conditioned media containingsuch factors may be used. Conditioned media may be obtained by culturingthe media with cells secreting such factors. Suitable cells includeirradiated (˜4,000 rad) primary mouse embryonic fibroblasts, telomerizedmouse fibroblasts, or fibroblast-like cells derived from primatepluripotent stem cells (U.S. Pat. No. 6,642,048). Medium can beconditioned by plating the feeders in a serum free medium such as KODMEM supplemented with 20% serum replacement and 4 ng/mL bFGF. Mediumthat has been conditioned for 1-2 days is supplemented with furtherbFGF, and used to support pPS cell culture for 1-2 days (see e.g., WO01/51616; Xu et al., (2001) Nat. Biotechnol. 19:971, 2001).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (like a fibroblast growth factor orforskolin) that promote proliferation of the cells in anundifferentiated form. Exemplary is a base medium like X-VIVO™ 10(Lonza, Walkersville, Md.) or QBSF™-60 (Quality Biological Inc.Gaithersburg, Md.), supplemented with bFGF at 40-80 ng/mL, andoptionally containing SCF (15 ng/mL), or Flt3 ligand (75 ng/mL) (see,e.g., Xu et al., (2005) Stem Cells 23(3):315). These medium formulationshave the advantage of supporting cell growth at 2-3 times the rate inother systems (see, e.g., WO 03/020920). In some embodiments pPS cellssuch as hES cells may be cultured in a media comprising bFGF and TGFβ.Suitable concentrations of bFGF include about 80 ng/ml. Suitableconcentrations of TGFβ include about 0.5 ng/ml. Other commerciallyavailable media formulations may be used in certain embodiments of theinvention. Suitable media formulations may include X-VIVO™ 15 (Lonza,Walkersville, Md.); mTeSR™ (Stem Cell Technologies, Vancouver, CA);hTeSR™ (Stem Cell Technologies, Vancouver, CA), StemPro™ (Invitrogen,Carlsbad, Calif.) and Cellgro™ DC (Mediatech, Inc., Manassas, Va.).

In some embodiments, the primate pluripotent stem cells may be platedat >15,000 cells cm⁻² (optimally 90,000 cm⁻² to 170,000 cm⁻²).Typically, enzymatic digestion may be halted before cells becomecompletely dispersed (e.g., about 5 minutes with collagenase IV). Clumpsof ˜10 to 2,000 cells may then be plated directly onto a suitablesubstrate without further dispersal. Alternatively, the cells may beharvested without enzymes before the plate reaches confluence byincubating the cells with for about 5 minutes in a solution of 0.5 mMEDTA in PBS or by simply detaching the desired cells from the platemechanically, such as by scraping or isolation with a fine pipette.After washing from the culture vessel, the cells may be plated into anew culture without further dispersal. In a further illustration,confluent human embryonic stem cells cultured in the absence of feedersmay be removed from the plates by incubating with a solution of 0.05%(wt/vol) trypsin (Gibco®, Carlsbad, Calif.) and 0.05 mM EDTA for 5-15min at 37° C. The remaining cells in the plate may be removed and thecells may be triturated into a suspension comprising single cells andsmall clusters, and then plated at densities of 50,000-200,000 cellscm⁻² to promote survival and limit differentiation.

In certain embodiments, pPS cells may be cultured on a layer of feedercells, typically fibroblasts derived from embryonic or fetal tissue(Thomson et al., (1998) Science 282:1145). In certain embodiments, thosefeeder cells may be derived from human or murine source. Human feedercells can be isolated from various human tissues or derived bydifferentiation of human embryonic stem cells into fibroblast cells(see, e.g., WO01/51616) In certain embodiments, human feeder cells thatmay be used include, but are not limited to, placental fibroblasts (see,e.g., Genbacev et al., (2005) Fertil. Steril. 83(5):1517), fallopiantube epithelial cells (see, e.g., Richards et al., 92002) Nat.Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al.,(2003) Biol. Reprod. 68:2150), uterine endometrial cells (see, e.g., Leeet al., (2005) Biol. Reprod. 72(1):42).

In the practice of the present invention, there are various solidsurfaces that may be used in the culturing of cells. Those solidsurfaces include, but are not limited to, standard commerciallyavailable cell culture plates such as 6-well, 24-well, 96-well, or144-well plates. Other solid surfaces include, but are not limited to,microcarriers and disks. In certain embodiments, the microcarriers arebeads. Those beads come in various forms such as Cytodex Dextranmicrocarrier beads with positive charge groups to augment cellattachment, gelatin/collagen-coated beads for cell attachment, andmacroporous microcarrier beads with different porosities for attachmentof cells. The Cytodex dextran, gelatin-coated and the macroporousmicrocarrier beads are commercially available (Sigma-Aldrich, St. Louis,Mo. or Solohill Engineering Inc., Ann Arbor, Mich.). In certainembodiments, the beads are 90-200 μm in size with an area of 350-500cm². Beads may be composed of a variety of materials such as, but notlimited to, glass or plastic. In certain embodiments, disks may be usedin stirred-tank bioreactors for attachment of the cells. Disks are soldby companies such as New Brunswick Scientific Co, Inc. (Edison, N.J.).In certain embodiments, the disks are Fibra-cel Disks, which arepolyester/polypropylene disks. A gram of these disks provide a surfacearea of 1200 cm².

The solid surface suitable for growing pPS cells may be made of avariety of substances including, but not limited to, glass or plasticsuch as polystyrene, polyvinylchloride, polycarbonate,polytetrafluorethylene, melinex, or thermanox. In certain embodiments ofthe invention, the solid surfaces may be three-dimensional in shape.Exemplary three-dimensional solid surfaces are described, e.g., inUS20050031598.

In certain embodiments, the cells are in a single-cell suspension duringthe methods of the invention. The single-cell suspension may beperformed in various ways including, but not limited to, culture in aspinner flask, in a shaker flask, or in a fermentors. Fermentors thatmay be used include, but are not limited to, Celligen Plus (NewBrunswick Scientific Co, Inc., Edison, N.J.), and the STR or theStirred-Tank Reactor (Applikon Inc., Foster City, Calif.). In certainembodiments, the bioreactors may be continuously perfused with media orused in a fed-batch mode. Bioreactors come in different sizes including2.2 L, 5 L, 7.5 L, 14 L or 20 L.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, and immunology.

With respect to tissue and cell culture and embryonic stem cells, thereader may wish to refer to any of numerous publications available inthe art, e.g., Teratocarcinomas and Embryonic Stem cells: A PracticalApproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al. eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R. I. Freshney,Culture of Animal Cells, Wiley-Liss, New York, 2000). With respect tothe biology of hematopoietic lineage cells the reader may refer to anyimmunology textbook, e.g., Immunobiology: The Immune System in Healthand Disease (Janeway et al., 2001 Garland Publishing).

Where derived from an established line of primate pluripotent stemcells, the cell populations and isolated cells of this invention can becharacterized as having the same genome as the line from which they arederived. This means that the chromosomal DNA will be essentiallyidentical by RFLP or by SNP analysis between the primate pluripotentstem cells and the differentiated progeny cells (assuming the cells havenot been genetically manipulated by the human hand). It is understoodthat minute alterations, e.g. in non-coding regions are possible;however the genetic identity between the line of pPS cells and therespective progeny will be comparable to that seen in identical twins.

Genetic Alteration of Differentiated Cells

The cells of this invention can be made to contain one or more geneticalterations by genetic engineering of the cells either before or afterdifferentiation (US 2002/0168766 A1). For example in some embodiments,the cells can be processed to increase their replication potential bygenetically altering the cells to express telomerase reversetranscriptase, either before or after they progress to restricteddevelopmental lineage cells or terminally differentiated cells (US2003/0022367 A1).

The cells of this invention can also be genetically altered in order toenhance their ability to be involved in modulating an immune response,or to deliver a therapeutic gene to a site of administration. A vectoris designed using the known encoding sequence for the desired gene,operatively linked to a promoter that is either pan-specific orspecifically active in the differentiated cell type. Alternatively thepromoter may be an inducible promoter that permits for the timedexpression of the genetic alteration. For example the cells may begenetically engineered to express a cytokine that modulates an immuneresponse either by enhancing the response or dampening the response.

In the following Examples all experiments utilizing human embryoniccells (hES) cells were performed using established hES cell lines.

EXAMPLES Example 1 Differentiation of hES Cells to mDC by Varying theDifferentiation Cocktail

In this example pPS cells were differentiated into mDC by firstculturing the pPS cells with a differentiation cocktail to obtain imDCand then further culturing the imDC with a maturation cocktail. Thedifferentiation cocktail comprised exogenous cytokines which varied overthe course of the experiment as the cells were differentiated to theimDC stage. (FIG. 1a ). Human ES cell line H1 (Thomson et al., (1998)Science 282:1145) were cultured feeder free in defined serum-free mediadevoid of animal-derived products (Xu et al., (2001) Nat Biotechnol19:971; Li et al., (2005) Biotechnol Bioeng 91:688) (FIG. 1b ). Thecells were also cultured stromal cell free throughout thedifferentiation and maturation protocol.

The hES cells were cultured under conditions permissive for formingembryoid bodies (EBs). Briefly, H1 cells were treated with collagenaseD, (Invitrogen, Carlsbad, Calif.) rinsed once with 1× PBS, and gentlyscraped off the plate with a cell scraper (Corning Life Sciences,Corning, N.Y.). The cells were then plated in 6 well ultra lowattachment plates (Corning Life Sciences, Corning, N.Y.) at 3 millioncells/well in X-VIVO 15 media (Lonzo, Walkersville, Md.) supplementedwith 1 mM Na-Pyruvate (Invitrogen, Carlsbad, Calif.), 1× non-essentialamino acids (Invitrogen, Carlsbad), Calif., 2 mM L-glutamine(Invitrogen, Carlsbad, Calif.), 5×10⁻⁵ M 2-Mercaptoethanol (Sigma, StLouis, Mo.), and 10 mM HEPES (Invitrogen, Carlsbad, Calif.) and allowedto form embryoid bodies. The following growth factors were added to themedium: SCF (20 ng/ml), VEGF (50 ng/ml), BMP-4 (50 ng/ml), and GM-CSF(50 ng/ml). All growth factors were purchased from R&D Systems (R&DSystems, Minneapolis Minn.). Each well contained 4 ml of media. Thecells were fed every two to three days with a 1:3 media change.

On day 5 BMP-4 was removed from the growth factor cocktail, on day 10VEGF was removed from the growth factor cocktail, and on day 15 SCF wasremoved from the growth factor cocktail (FIG. 1A). At around d17-d25round shining hematopoietic progenitor cells were visible (FIG. 2). Whenabout 100,000 to about 1 million floating shining progenitor cells werevisible in the wells, they were harvested, spun down and reseeded in theoriginal 6 well ultra low attachment plates and cultured in X-VIVO 15(Lonzo, Walkersville, Md.) with GM-CSF (50 ng/ml) and IL-4 (50 ng/ml)(R&D Systems, Minneapolis, Minn.) to generate imDC. The original EBswere moved to new 6 well ultra low attachment plates for about 40-50days and fed with media comprising GM-CSF (50 ng/ml) every 2-3 days (1:3media change) and continued to produce shiny hematopoietic cells whichwere harvested and then further cultured and differentiated with GM-CSFand IL-4 to produce additional imDC.

Flow cytometric analysis on the hES cells was performed as follows: thecells were resuspended in 50 μl of Flow buffer (PBS+0.1% BSA+2 mM EDTA)and blocked using an anti-FC receptor antibody (Miltenyi, Aurburn,Calif.) for 10 minutes at 4° C. and then antibodies to the target markerwere added (antibodies are provided below in Table I). After incubationfor 20 minutes at 4° C., the cells were washed 2× in Flow buffer, and 5minutes before sample analysis 2 ul 7AAD (0.25 ug/1×10⁶ cells) (BDBioscience, San Jose, Calif.) was added per sample in order to assesscell viability. Sample data were collected using a FACSCalibur™ (BectonDickinson, San Jose, Calif.), and analyzed using FlowJo® software(Treestar, Ashland, Oreg.). For intracellular Oct-4 staining the cellswere fixed using an intracellular fixation buffer (eBioscience, SanDiego, Calif.) and made permeable using a permeabilization buffer(eBioscience, San Diego, Calif.) according to the manufacturer'sinstructions. As expected the cells expressed Oct-4 and SSEA-4 bothmarkers of hES cells. In addition the cells also expressed Flt-1 andFlk-1 both receptors for VEGF, as well as CD117, the receptor for SCF.CD116, the GM-CSF receptor, was not detected (FIG. 1C).

The imDC were analyzed by flow cytometry (as described above) for thefollowing markers: CD14, HLA-I, HLA-II, CD83, CD205 and CD11b. The cellswere found to be positive for HLA-I, HLA-II, CD83, and CD11b (FIG. 4A).After 4-6 days these immature DC were spun down and resuspended inX-VIVO 15 media comprising the following cytokines IFN-γ (25 ng/ml),IL-1-β (10 ng/ml), TNF-α (10 ng/ml), PGE2 (1 μg/ml) and GM-CSF (50ng/ml) (the maturation cocktail). The cells were maintained in culturefor an additional 48 hours to generate matured DC. The mDC were analyzedby FACs (as described above) and found to express the following markers:HLA-I, HLA-II, CD40, CD86, CD83, CD205, CD11c^(hi), and CCR7. The cellswere negative for CD14 (FIG. 4B). CD83 is a marker for dendritic cellmaturation. CCR7 is a chemokine receptor involved in DC migration. Theexpression profile was comparable to DC derived from peripheral bloodmononuclear cells (PBMC). The cells were also analyzed by real timequantitative PCR for expression of the following transcription factors:NF-κB, CIITA and Spi-B (FIG. 4C). Spi-B has been shown to be expressedin DC derived from PBMCs (Schotte et al., (2003) Blood 101(3):1015;Rissoan et al., (2002) Blood 100(9):3295; Schotte et al., (2004) J. Exp.Med. 200(11):1503; Chicha et al., (2004) J. Exp. Med. 200(11):1519.NF-κB is associated with costimulatory molecule expression and isnecessary for the DC activation process. CIITA is a master regulator ofHLA-II expression.

The cells were analyzed morphologically and were found to have amorphology typical of DC (FIG. 4D). To further study the morphology ofthe mDC the cells were stained with May-Grunwald stain. The cells werewashed with 1× PBS, resuspended in 50 ul of 1× PBS, added onto glassslides prepared with the cytospin apparatus. Ten ul of the cellsuspension was applied to the slide. The slides were spun at 1200 rpmfor 5 minutes using a ShandonCytospin3 (Thermo Scientific, WalthamMass.). The cells were then stained with May Grunwald Staining solution(Sigma, St Louis, Mo.) for 5 minutes at 25° C., washed 3× with dH₂O, andair dried overnight. The sample area was coated with Permount® (Sigma,St Louis, Mo.), a glass cover slip was applied, and the slide wasallowed to dry overnight. Images were taken with an upright microscopewith 100× planar objective and 40× Plan-Neofluar objective (Zeiss,Peabody, Mass.). The results shown in FIG. 4E demonstrated that the mDChad morphology typical of a dendritic cell isolated from PBMCs thatincluded branched projections or dendrites emanating out from the cell.The cells were also analyzed by flow cytometry (as described above) forCD19, CD3, CD235a and CD41 (indicative of B cells, T cells,erythrocytes, and platelets, megakaryocytes, respectively), but werefound to be undetectable for all of these markers. The preparation wasfound to have 5-20% granulocytes and 5-20% progenitor cells. Thepopulation of cells ranged from about 50% to about 90% DC.

Example 2 Time Course Analysis of Differentiating Cell Cultures

In order to characterize precursor cell populations arising during theprocess of differentiating pPS to imDC, the cell culture described inExample 1 was evaluated over time for transcription factor expressionusing RT PCR and real time quantitative PCR (as described below) and byflow cytometry (as described above) over time for expression of cellsurface markers.

For real time quantitative PCR target cells were harvested and total RNAwas isolated following the standard Qiagen RNeasy® Mini Prep protocol(Qiagen, Valencia, Calif.). Qiagen QiaShredder (Qiagen, Valencia,Calif.) was used to homogenize the lysate. Isolated RNAs were stored at−80° C. For cDNA synthesis, 1 μg RNA samples were treated with DNase(Ambion, Austin Tex.) to remove any genomic DNA impurities from the RNAprep. Reverse Transcriptase PCR (RT-PCR) was performed using SuperscriptII™ (Invitrogen, Carlsbad, Calif.) first strand synthesis system to makecDNA. The cDNA product was diluted 1:5 in water and used as a templatefor the Taqman® PCR Cycle Threshold (CT) Real Time Quantitation (AppliedBiosystems, Foster City, Calif.). Samples were run on Applied BioSystems7900HT Sequence Detection System (Applied Biosystems, Foster City,Calif.). The data were analyzed for relative expression level bynormalizing the target signal against the target signal at day 0. FIGS.3a and 3b provide the results. By day 5 expression of Oct-4 decreased 20fold. The five fold increase of brachyury by day 5 indicated the cellshad differentiated into mesoderm. Flk-1 is found on hematopoietic stemcells. The increased expression of Flk-1 suggested the differentiationof a hemogenic cell population. The expression of Tie-2 suggested thedifferentiation of hemangioblasts. Hemangioblasts are comprised of cellswith both hematopoietic and endothelial potential.

FIG. 3b shows expression levels of additional transcription factors overtime. Increased expression levels of both HoxB4 and Gata2 suggested thatthe cells have differentiated into a hematopoietic cell population.HoxB4 plays a role in hematopoietic stem cell renewal and survival(Antonchuk et al., (2002) Cell 109(1):39. Gata2 is an earlyhematopoietic transcription factor that is also expressed in GMP.

Flow cytometry was performed as described in Example 1. Antibodies usedin all flow cytometry experiments are listed below in Table I.

TABLE I SSEA-4 R&D Systems 4-Oct Santa Cruz Biotechnology CD117 BDBioscience Flt-1 R&D Systems RD-KDR R&D Systems CD116 BD Bioscience CD19BD Bioscience CD3 eBioscience CD11b BD Bioscience CD11c BD BioscienceCD13 BD Bioscience CD15 BD Bioscience CD34 BD Bioscience CD38 BDBioscience CD40 BD Bioscience CD44 BD Bioscience CD45 BD Bioscience CD80BD Bioscience CD86 BD Bioscience CD83 BD Bioscience HLA-I BD BioscienceHLA-II BD Bioscience CD205 BD Bioscience CD303 Miltenyi CD123 BDBioscience CCR7 BD Bioscience

FIGS. 3c and 3d shows the results of the flow cytometric study forexpression of CD45 and CD34 over time. The expression of CD34 by day 5was indicative of early hematopoiesis. By day 5 the morphology of theculture had taken on the appearance of a cystic embryoid body (FIG. 3e). By day 15 expression of CD45, a pan-hematopoietic cell marker, wasapparent. At the same time transcription factor PU.1 was detected byreal time quantitative PCR (FIG. 3b ). PU.1 expression is found in earlyhematopoietic cells and its expression level increases as the cellsdifferentiate to dendritic cells (Guerriero et al., (2000) Blood95(3):879; Nutt et al., (2005 J Exp. Med. 201(2):221). Myeloid lineagemarker CD13 expression became apparent by day 15 (FIG. 3F). Thissuggested that by the time SCF was removed from the differentiationcocktail the cells had already entered the hematopoietic and myeloidlineage. Monocyte marker CD14 was expressed by day 20 (FIG. 3F).Expression of both CD13 and CD14 increased with time (FIG. 3F).

By day 20 it became apparent that there were two CD45+ populations inthe culture: CD45^(hi) and CD45^(lo) (FIG. 3G). The expression of CD14increased over time in the CD45^(hi) population. By day 32 65% of thecells expressed CD14 and CD45. CD14 expression was not seen in theCD45^(lo) population (FIG. 3G). The CD45^(hi) population correlated withcells in the monocyte/dendritic cell gate named (R1) while the CD45^(lo)population correlated with the granulocyte progenitor cell gate named(R2) in the forward scatter versus side scatter plot (FIG. 3G). TheCD45^(hi) population was characterized further at day 32 of thedifferentiation protocol and found to be positive for CD11c, CD11b,HLA-I, HLA-II^(lo/neg) and CD86 (FIG. 3H) all suggesting the cells wereimDC. CD86 is a costimulatory molecule involved in T cell activation,while CD83 is mDC marker. Lack of CD83 expression indicated the cellswere not mDC.

Example 3 Antigen Processing and Presentation

To test the ability of the hES derived imDC to process antigen, thefluorescent dye, DQ-OVA (Invitrogen, Carlsbad, Calif.), was dissolved at1 mg/ml in PBS and added at 100 μg/ml to imDC derived from hES asdescribed in Example 1. The protein was labeled with a pH insensitiveBODIPY-F1 dye. The dye is self quenching when the protein is intact, butfluoresces bright green when the protein is denatured or undergoesproteolysis. The cells were incubated either at 37° C. or at 4° C. (as acontrol for background fluorescence) and washed 2× with flow buffer.Data was collected with FACSCalibur™ (Becton Dickinson, San Jose,Calif.) in FL1. The treated cells were found to fluoresce indicatingthat the protein had been proteolyzed by the imDC, while the controlcells did not (FIG. 5a ).

A functional assay was performed next to determine if DC made accordingto the method of Example 1 were able to stimulate antigen specificlymphocyte secretion of IFNγ, one hallmark of the adaptive immuneresponse. Mumps protein was used as the stimulatory antigen (Biodesign,Saco, Me.). The protein was added at 100 ug/ml for 1 hour to imDCderived from hES as described in Example 1. The maturation cocktaildescribed earlier IFN-γ (25 ng/ml), IL-1-β (10 ng/ml), TNF-α (10 ng/ml),PGE2 (1 μg/ml) and GM-CSF (50 ng/ml) was added next. After 24 hours,matured DC, either untreated or treated with mumps protein, werecollected, and washed 2× with AIM-V media (Invitrogen, Carlsbad,Calif.). The DC were plated at 1×10⁴ cells/well together with 1×10⁵PBMCs/well (Cellular Technologies LTD, Shaker Heights, Ohio) IFNγELISPOT plates were used for the read out (Millipore Corp. Bedford,Mass.). ELISPOT plates were coated with anti-IFNγ Ab (Mabtech,Mariemont, Ohio) at 10 ug/ml overnight (16-24 hours). The assay platewas placed at 37° C. and 5% CO₂ for 16-24 hours, and developed followingthe instructions provided by Mabtech. Spots were counted using a CTLAnalyzer (Cellular Technology Limited, Decatur, Ill.). The resultspresented in FIG. 5B demonstrate a 3 fold difference in IFNγ productionby mDC of the invention over the non-treated control.

Example 4 Cytokine Production

A qualitative cytokine array analysis was performed using the HumanCytokine Array III and V kit (Raybiotech, Norcross, Ga.) on both imDCand mDC obtained according to the method described in Example 1. Theassay was performed according to the manufacturer's instructions. ThemDC were found to produce the following pro-inflammatory cytokines:IL-6, IL-7, IL-8, and Il-10. IL-7 is believed to be important for T cellsurvival. IL-8 is believed to be a chemotactic stimulus. Cytokines IL-6,IL-10, and IL-12 were quantified using the BD Cytometric Bead Array (BDBiosciences, San Jose, Calif.) following the manufacturers instructions.Supernatants from immature and mature DC derived from hES were collectedand concentrated using Amicon Ultra-15 10,000 NMWL (Millipore, Bedford,Mass.) centrifuge tubes. The supernatants were added to human IL-6,IL-10, and IL-12 bead flex sets (BD Biosciences, San Jose, Calif.), andincubated for 1 hour at 25° C. Antibody detection reagent conjugated toPE (BD Biosciences, San Jose, Calif.) was added and incubated for anadditional 2 hours at room temperature. Samples were washed 1×,re-suspended in wash buffer (BD Biosciences, San Jose, Calif.), andcollected by flow cytometry with a FACSCaliber™ (Becton Dickinson, SanJose, Calif.). Cytokine concentrations were determined using FCAP ArraySoftware (BD Biosciences, San Jose Calif.). The results presented inFIG. 6A demonstrate significant levels of all three cytokines wereproduced by the mDC.

Example 5 Chemotactic Analysis of mDC

AIM-V media (Invitrogen, Carlsbad, Calif.) was added to the upper andlower chambers of Transwell 24 well plates containing 8.0 uM pore sizeinserts (Corning, Corning, N.Y.), and incubated overnight at 37° C., 5%CO₂. After removal of the media from each well, 0.6 ml of AIM-V with orwithout the chemokine MIP3β (100 ng/ml) was added to the lower chamber.Mature DC derived from hES (as described in Example 1) were harvestedand washed 2× in AIM-V media. The cells were resuspended in AIM-V mediaat 2.0×10⁶ cells/ml, and 0.1 ml was added to the top chamber. Thetranswell plate was incubated for 2 hours at 37° C., 5% CO₂. The numberof cells that migrated to the bottom chamber was determined using ahemacytometer. The results presented in FIG. 6D demonstrated that mDCaccording to the invention migrate in response to MIP3β.

Example 6 Immunostimulatory Capacity of Dendritic Cells

Several assays were performed to characterize the ability of mDCproduced according to the method of Example 1 to stimulate an immuneresponse. First a mixed lymphocyte reaction (MLR) assay was done todemonstrate that the mDC had the ability to stimulate a strong naïveallo-response.

PBMC derived DC were prepared by isolating peripheral blood mononuclearcells (PBMCs) from fresh healthy donor buffy coat preps. PBMCs wereadhered to tissue culture flasks in AIM-V media for 2 hours then washedwith warm PBS to remove non-adherent cells. The remaining adherentcells, comprised mostly of monocytes, were incubated at 37° C. and 5%CO₂ for 6 days with recombinant human interleukin 4 (rhIL-4) (R&DSystems, Minneapolis Minn.) and recombinant human GM-CSF (rhGM-CSF) (R&DSystems, Minneapolis Minn.) at 1000 U/ml to generate imDC. imDC werethen matured for 24 hours in AIM-V media (Invitrogen, Carlsbad, Calif.)with 800 U/ml rhGM-CSF, 10 ng/ml TNFα, 10 ng/ml IL1-β, and 10 ng/ml IL-6and 1.0 ug/ml PGE2 (R&D Systems, Minneapolis Minn.).

For MLR assay PBMCs were isolated from buffy coats obtained from healthyvolunteers (Stanford Blood Bank) by centrifuging the cells over aFicoll-Paque gradient (Amersham Pharmacia Biotech AB, Buckinghamshire,UK). Isolated cells were washed and resuspended in complete RPMI 1640medium (Invitrogen, Carlsbad, Calif.) with 10% FBS (Clonetech, MountainView, Calif.) and 1% penicillin/streptomycin (Invitrogen, Carlsbad,Calif.). In a 96-well U-bottom plate (Becton Dickinson, San Jose,Calif.), 5×10⁴ PBMCs and different numbers of irradiated stimulatorcells (2000 rads for hESCs, monocyte derived DC and hES derived DC) weremixed in a 96-well U-bottom plate (Becton Dickinson, San Jose, Calif.)and incubated at 5% CO₂ and 37° C. for five days. The cells were thenpulsed with 1 uCi ³H thymidine per well for 18 hours at 5% CO₂ and 37°C. The cells were harvested onto a UniFilter-96 GF/C (PerkinElmer,Waltham, Mass.) using a Filtermate Harvester (Perkin Elmer, Waltham,Mass.) and ³H thymidine incorporation was counted using a TopCountscintillation counter (Perkin Elmer, Waltham, Mass.). The resultsdemonstrated that the mDC produced according to Example 1 had goodallogeneic stimulating activity (FIG. 7A).

Next the ability of the mDC to stimulate antigen specific effector Tcells was investigated. CMV peptide pp65 (amino acid sequence 495-503)was used to demonstrate DC antigen presentation to CD8+ lymphocytes.Characterized PBMC responders comprising CD8 lymphocytes, whichspecifically recognize the pp65 peptide were used. The CD8 T-lymphocytesand the DC shared a common HLA-A2 allele. For CMV specific antigenpresentation, matured PBMC-DC and hES derived DC were resuspended in 150ul of serum free AIM-V medium (Invitrogen, Carlsbad, Calif.) eithersupplemented with 10 μg/ml CMV pp65 peptide or unsupplemented. The cellswere incubated at 37° C. and 5% CO₂ for 2 hours, and then washed 2× withAIM-V media (Invitrogen, Carlsbad, Calif.). DC were plated onto anELISPOT plate at 1×10⁴ cells/100 ul/well at a 10:1 responder tostimulator ratio. Characterized PBMC responders, specific for CMV,(Cellular Technologies Limited, Decatur, Ill.) were thawed in 37° C.water bath, washed two times, resuspended in AIM-V media (Invitrogen,Carlsbad, Calif.), and plated at 1×10⁵ cells/100 ul/well on ELISPOTplates (Millipore, Bedford Mass.). ELISPOT plates were coated withanti-IFNγ Ab (Mabtech, Mariemont, Ohio) at 10 ug/ml overnight (16-24hours). The assay plate was placed at 37° C. and 5% CO₂ for 16-24 hours,and developed following the instructions provided by Mabtech. Spots werecounted using a CTL Analyzer (Cellular Technology Limited, Decatur,Ill.). The results, shown in FIG. 7b , demonstrated that mDC (labeledES-DC in the figure) were able to stimulate IFNγ production that wascomparable to PBMC-DC.

Next the ability of mDC to stimulate T cell expansion in vitro wasexamined. A CMV T cell line (67% specificity) (ProImmune, Bradenton,Fla.) was thaw at 37° C. and washed 2× with 1640 RPMI medium(Invitrogen, Carlsbad, Calif.)+5% FBS (Invitrogen, Carlsbad, Calif.)supplemented with 1 mM Na-Pyruvate, non-essential amino acids, 2 mML-glutamine, 5×10⁻⁵ M 2-Mercaptoethanol, and HEPES. The T cells werethen incubated at 37° C. and 5% CO₂ for 2 hours. 5 mM CellTrace CFSEstock solution (Invitrogen, Carlsbad, Calif.) was dissolved immediatelyprior to use in DMSO (Invitrogen, Carlsbad, Calif.). The T cells wereresuspended in pre-warmed PBS/0.1% BSA at 1×10⁶/ml. The dye was added tothe cells at a final concentration of 2 uM and incubated at 37° C. for10 minutes. The Celltrace dye was quenched by the addition of 5×ice-cold culture media. The cells were washed 2× before setting up theassay.

DC derived from hES were prepulsed with 10 μg/ml of CMV495-503 pp65peptide (Anaspec, San Jose, Calif.) (>95% pure by HPLC) for 2 hours at37° C. and washed 2× before plating at 2×10⁴/well in 96 well U bottomFalcon™ plate (BD, San Jose, Calif.). CFSE labeled T cells were platedat 2×10⁵ cells/well. On day 5, cells were harvested and stained with 5μl of CMV495-503 specific pentamer labeled with APC (ProImmune,Bradenton, Fla.) per million cells at 25° C. for 10 minutes. Thisrecognized T cells specific for the CMV peptide 495-503. This permittedgating the FACS on this specific population of cells for the CFSEanalysis. The cells were washed 2× with flow buffer, stained with 7AADfor 5 minutes before running the samples on the FACSCalibur™ (BectonDickinson, San Jose, Calif.). Dead cells were excluded from analysis by7AAD. Dilution of the dye label (multiple peaks) is indicative of T cellproliferation. As shown in FIG. 7C the DC made according to Example 1(ES-DC in the figure) caused T cell proliferation comparable to PBMC-DC.The CD8 T lymphocytes and the dendritic cells shared a common HLA-A2allele.

Example 7 Comparison of Differentiation Cocktails

The culture conditions for growing and differentiating hES cells to imDCand mDC was performed as described in Example 1 except that thedifferentiation cocktail used was changed to compare differentiationcocktails comprising a variety of exogenous cytokines. Variouscombinations of 7, 5, 4 and 3 cytokines (growth factors) were tested fortheir ability to differentiate hES cells to imDC. Table II providesdetails regarding experiments in which the differentiation cocktailcomprised 7, 5, and 4 exogenous cytokines (plus signs indicate thepresence of the factor/minus signs indicate the factor was not used) Thenumbers in the bottom half of the table indicate the percentages of eachcell marker obtained with the corresponding cocktail indicated directlyabove the percentages. Table III provides details in which thedifferentiation cocktail comprised 4 and 3 exogenous cytokines (plussigns indicate the presence of the factor/minus signs indicate thefactor was not used). The setup regarding percentages of markersobtained relative to the corresponding cocktails is indicated in thebottom half of the table where the percentages correspond to thecocktail shown above the numerical data. Tables IV-VI provide detailsregarding the composition of the differentiation cocktail (as describedin Tables II and III) over the time course of the experiment (“X”sindicate the factor was present for the specified period) (“d” is usedin these tables as an abbreviation for “day”).

imDC were matured to mDC using two different maturation cocktails. Inthe experiments described in Table II a maturation cocktail comprisingGM-CSF, Il-1β, IFN-γ, CD40L and IFNα was used. In the experimentsdescribed in Table III a maturation cocktail comprising TNFα, IL1β, IFNγand PGE2 was used. Table VII provides the concentration and source ofeach of the cytokines (growth factors) tested.

TABLE II Growth Factor Reduction Experiment BMP-4 + + + GM-CSF + + +VEGF + + + SCF + + + Flt3-L + + − TPO + − − IL-3 + − − Time Marker %positive d 20 CD45 67.2 ± 6.71 72.1 ± 5.2  71.2 ± 7.46 CD11c 39.2 ± 6.4942.9 ± 6.33 36.7 ± 7.23 CD14 23.1 ± 8.15 23.7 ± 8.4  25.3 ± 8.7  d 30CD45 89.6 ± 1.29 91.7 ± 0.73 86.5 ± 2.94 CD11c 82.7 ± 1.67 83.1 ± 1.3972.5 ± 4.61 CD14 22.2 ± 3.74 25.4 ± 4.56 26.8 ± 4.15 iDC CD86 65.5 ±3.9  67.4 ± 4.29 71.7 ± 3.02 CD83 37.8 ± 1.74 47.6 ± 2.55 45.3 ± 3.51MHC II 34.2 ± 7.5   31 ± 4.78 25.6 ± 6.03 mDC CD86 63.9 ± 4.51 73.4 ±4.48 74.9 ± 2.58 CD83 63.3 ± 3.8  69.8 ± 5.09  71 ± 3.83 MHC II 43.3 ±5.23 46.6 ± 6.08 34.5 ± 7.26 CCR7 44.7 ± 5.32 56.7 ± 6.41  57 ± 4.79yield^(#) 10³ cells/well* 335 ± 92 385 ± 7.1 666 ± 182 % cells positiveof total population. Average is n = 4 with mean standard error.*Differentiations were done in ultra low attachment 6 well plates.^(#)average of n = 2.

TABLE III Growth Factor Reduction Experiment BMP-4 + − + + + GM-CSF + +− + + VEGF + + + − + SCF + + + + − Time Marker % positive d 20 CD34 26.511.3 1.3 ± 0.17  14 ± 9.5 20.8 ± 16.5 18.4 ± 11.4 CD45 76.4 10.9 1.2 ±0.54 7.6 ± 6.4 30.1 ± 27.2 54.2 ± 25.7 CD11c 42.1 20.8 1.5 ± 0.74 1.9 ±0.4 4.2 ± 2.7 27.8 ± 23.4 d 30 CD45 70.7 10.8 0.7 ± 0.4   12 ± 7.1 51 ±23 77.3 ± 6   CD11c 69.6 5.9 0.7 ± 0.4   9 ± 4.5 46.7 ± 21.4 65.4 ± 5.9 CD14 23 5.8 0.8 ± 0.6  8.3 ± 5  20.8 ± 11  27.6 ± 7.9  % cells positiveof total population. *Differentiations were done in ultra low attachment6 well plates Average is n = 3 with mean standard error

TABLE IV 7 growth factors BMP-4 IL-3 VEGF TPO SCF flt3L GM-CSF d 0-5 x xx x x x x d 6-10 x x x x x d 11-15 x x x d 16 on x x

TABLE V 5 growth factors 4 growth factors BMP-4 VEGF SCF flt3L GM-CSFBMP-4 VEGF SCF GM-CSF d 0-5 x x x x x x x x x d 6-10 x x x x x x X d11-15 x x x x x d 16 on x x x

TABLE VI 3 growth factors 3 growth factors 3 growth factors 3 growthfactors BMP-4 VEGF GM-CSF BMP-4 SCF GM-CSF BMP-4 VEGF SCF VEGF SCFGM-CSF d 0-5 x x x x x x x x x x x x d 6-10 x x x x x x x x x d 11-15 xx x x x x d 16 on x x x

TABLE VII Reagents Growth Factors Manufacture Catalogue ConcentrationUsed rhBMP-4 R&D Systems 314-BP 50 ng/ml rhSCF R&D Systems 255-SC-050 20ng/ml rhGM-CSF R&D Systems 215-GM-050 50 ng/ml rhFLT3L R&D Systems308-FKN-025 20 ng/ml rhVEGF R&D Systems 293-VE-050 50 ng/ml rhIL-4 R&DSystems 204-IL-010 50 ng/ml rhTNF-alpha R&D Systems 210-TA-010 10 ng/mlrhIFN-gamma R&D Systems 285-IF-100 20 ng/ml rhIL-3 R&D Systems203-IL-050 25 ng/ml MIP-3B Pepro Tech 300-29B 100 ng/ml rhCD40L R&DSystems 617-CL-050/CF 100 ng/ml rhIFN-alpha R&D Systems 11101-2 10 ng/mlIL-1beta R&D Systems 201-LB-005 10 ng/ml

Example 8 Comparison of Maturation Cocktail

pPS cells differentiated to imDC according to various embodiments of theinvention were matured to mDC using different combinations ofcytokines/factors. A cell concentration of 0.05×10⁶ cells/well wereplated in 96 well plates and cultured for 24 hrs in X VIVO-15 mediasupplemented with various combinations of cytokines/factors as set forthin Table VIII. Concentrations of the factors used were as set forthabove in Table VII. For Poly I:C 10 ug/ml was used; for PGE2 1 ug/ml wasused; for iL-6 10 ng/ml was used. All tested maturation cocktailscontained 50 ng/ml of GM-CSF. IL-12 and IL-10 levels from supernatantsat 24 hours was measured using the BD™ Cytometric Bead Array (BDBiosciences, Franklin Lakes, N.J.) as indicative of maturation of imDCto mDC. The results suggested that as few as four exogenous cytokinescould stimulate the maturation of imDC to mDC.

TABLE VIII Maturation cocktail pg/ml TNFα IL1β IFNγ PGE2 POLY I:C IFNαCD40L IL-12 IL-10 1 − − − − − − − 0.0 35.8 2 + + + + − − − 1.9 118.43 + + + + − − + 2.6 104.8 4 + + + + + + − 1.4 219.8 5 + + + + + + + 1.9206.3 6 + + + − + + − 0.0 155.6 7 + + + − + + + 1.9 202.8

The experiment was repeated using a cell concentration of 0.2×10⁶cells/well in 6 well plates, but this time a different panel ofcytokines was tested. The cytokine concentrations were as follows: TNFα10 ng/ml; IL-1β 10 ng/ml; IFN γ 20 ng/ml PGE2 1 ug/ml; IL-6 10 ng/ml.The supernatants were concentrated using Amicon Ultra-15 10,000 NMWLcentrifuge tubes (Millipore Corp, Bedford, Mass.) and analyzed after 48hour exposure to the various maturation cocktails for IL-12 and IL-6production as indicative of DC maturation. As in the previous experimentall maturation cocktails also contained 50 ng/ml of GM-CSF. Alsoincluded as a positive control were monocyte-derived DC generated fromhuman PBMCs. The results are presented in Table IX below.

TABLE IX Maturation Cocktail Cytokine (pg/ml) IFN-γ TNF-α IL-1β PGE2IL-6 IL-12 IL-6 Cell type − − − − − 2.0 96.3 es-iDCs + + + + − 11.638,729.2 es-mDCs − + + + + 2.2 53,438.0 es-mDCs + − + + − 3.4 9,796.6es-mDCs + + − + − 1.7 195.3 es-mDCs − + + + + 16.5 64,856.8 Mo-mDCs

Example 9 Generation of hTERT T Cell Lines

PBMCs were isolated from HLA-A2+ buffy coats of healthy human donorsusing Ficoll Plaque-Plus (GE Healthcare Bioscience AB, Piscataway, N.J.)separation methods. To generate imDC, monocytes from HLA-A2+ PBMCs wereisolated using CD14+ microbeads (Miltenyi, Aurburn, Calif.), andtransferred into serum free AIM-V media (Invitrogen, Carlsbad, Calif.)containing rhGM-CSF (1000 U/ml) (Berlex, Richmond, Calif.) and rhIL-4(1000 U/ml) (R&D systems, Minneapolis, Minn.), and incubated at 37° C.5% CO₂ for 5 days. DC were matured for 24 hours by adding a cytokinecocktail comprising TNFα (10 ng/ml) (R&D systems), IL1β (10 ng/ml) (R&Dsystems), IL-6 (10 ng/ml) (R&D systems), and PGE2 (1 ug/ml) (R&Dsystems). mDC were harvested, washed 2× in AIM-V media, resuspended in200 ul of AIM-V media, and pulsed with 540 hTERT peptide, a 9merbeginning with amino acid 540 of the hTERT protein (100 ug/ml) (AnaSpecInc, San Jose, Calif.) for 2 hours at 37° C. 5% CO₂.

Autologous CD8+ T cells were isolated from PBMCs by depleting non-CD8+cells using a CD8+ T cell magnetic separation kit (Miltenyi, Aurburn,Calif.). CD8+ cells were resuspended in AIM-V media containing 10% humanAB serum (Valley Biomedical, Winchester, Va.) and transferred to 24wells plates at a concentration of 1.0-2.0×10⁶ cells/ml. 540 hTERTpulsed DC were added to the wells at a stimulator to responder ratio of1:10 and incubated at 37° C. 5% CO₂. The following day, recombinanthuman IL-7 (10 ng/ml) (R&D systems) and IL-2 (10 U/ml) (R&D systems)were added to the culture.

Peptide hTERT 540 restimulations were performed every 7-10 days. Forrestimulation autologous PBMC were used to present the antigen.Autologous PBMCs were transferred to 24 well plates at a concentrationof 2.0-3.0×10⁶ cells/well containing serum free AIM-V media and hTERT540 peptide (10 ug/ml). PBMCs were kept at 37° C. 5% CO₂ for 2 hours topromote attachment of cells to the plate. Non-adherent cells wereremoved with 2× washes with AIM-V media. Adherent PBMCs were pulsed foran additional 2 hours with 540 hTERT peptide (10 ug/ml) and thenirradiated at 2000 rads.

CD8+ T cells from the initial priming with 540 hTERT pulsed DC wereharvested, washed 1×, and transferred into wells containing 540 hTERTpulsed irradiated adherent PBMCs. IL-12 (10 ng/ml) (R&D systems) wasadded to the culture. The following day, recombinant human IL-7 (10ng/ml) and/or IL-2 (10 U/ml) were added to the culture. Every 3-4 days,half the medium was removed and fresh medium was added containing IL-7and/or IL-2 when appropriate. At least 3 restimulations using 540 hTERTpulsed irradiated autologous adherent PBMCs were performed.

The percent positive 540 hTERT specific CD8+ T cells were determined bystaining cells with 540 pentamer labeled with APC (ProImmune, Bradenton,Fla.) and anti-human CD8 FITC conjugated Ab (Proimmune, Bradenton, Fla.)using FlowJo software (Tree Star, Ashland, Oreg.). The TERT specificCD8+ cells were collected by flow cytometry using a FACSCaliber™ (BectonDickinson, San Jose, Calif.) and used in subsequent experiments.

Example 10 ELISpot IFNγ Assay of 540 hTERT T Cell Lines

hES derived mDC (Example 1) were resuspended in 200 ul of serum freeAIM-V media (Invitrogen, Carlsbad, Calif.) and pulsed with 540 hTERTpeptide (100 ug/ml) for 2 hours at 37° C. 5% CO₂. Non-pulsed mDC servedas a control. Non-pulsed hES derived mDC served as a control. The mDCwere washed 2× in AIM-V media, resuspended in AIM-V media, and platedwith 540 hTERT T cell lines at a stimulator to responder ratio of 1:10in ELISpot plates coated with anti-IFN-γ Ab (10 ug/ml) (Mabtech,Mariemont, Ohio). The assay plate was placed at 37° C. 5% CO₂ for 16-24hours, and developed following the instructions provided by themanufacturer. Spots were counted using a CTL Analyzer (CellularTechnology Limited, Decatur, Ill.). The results are shown in FIG. 8 anddemonstrate that mDC differentiated from hES stimulate a specific T cellresponse to a hTERT antigen.

Example 11 Proliferation of 540 hTERT T Cell Lines

The 540 hTERT T cell lines were resuspended in pre-warmed PBS/0.1% BSAat 1.0×10⁶ /ml. CFSE (Invitrogen, Carlsbad, Calif.) was added to thecells at a final concentration of 2 uM and incubated at 37° C. for 10minutes. The stain was quenched by the addition of prechilled AIM-Vmedia containing 10% PBS (Clonetech, Mountain View, Calif.). The cellswere washed 2× before setting up the assay. Mature hES derived DC(Example 1) were pulsed with 10 μg/ml of 540 hTERT peptide (Anaspec, SanJose, Calif.) for 2 hours at 37° C. 5% CO₂ and washed 2× in AIM-V mediabefore plating at 2×10⁴/well in 96 well U bottom Falcon™ plate (BD, SanJose, Calif.). CFSE labeled 540 hTERT T cell lines were plated at 2×10⁵cells/well. Non-pulsed hES derived mDC served as a control. On day 4,cells were harvested and stained with 540 pentamer reagent conjugated toAPC (ProImmune, Bradenton, Fla.). The cells were washed 2× with FACSbuffer, and stained with 7AAD prior to collection using a FACSCaliber™(Becton Dickinson, San Jose, Calif.). The analysis was performed usingFlowJo software (Tree Star, Ashland, Oreg.). The results are presentedin FIG. 9 and demonstrate that mDC differentiated from hES cells canpresent hTERT antigen in the context of HLA-A2 and stimulate antigenspecific T cell proliferation.

Example 12 Immunostimulatory Capacity of Irradiated mDC

Mature dendritic cells were differentiated in vitro from pPS cellsaccording to the method described in Example 1. To address the effectsof irradiation on dendritic cells differentiated in vitro from hES cells(hESC-DCs), the ability of irradiated and non-irradiated hESC-DCs tostimulate antigen specific effector responses of T cells was compared.CMV peptide pp65 (amino acid sequence 495-503) was used to demonstratehESC-DC antigen presentation to CD8+ lymphocytes. Characterized PBMCresponders (Cellular Technology Limited, Decatur, Ill.), which containCD8+ T cells that recognize pp65 complexed to HLA-A2 were used as theresponder cells. For the pp65 specific antigen presentation, maturedhESC-DC were resuspended in 150 ul of serum free AIM-V medium(Invitrogen, Carlsbad, Calif.) either supplemented with 10 μg/ml pp65peptide or unsupplemented. The cells were incubated at 37° C. in 5% CO₂for 2 hours, and then washed 2 times with AIM-V media (Invitrogen,Carlsbad, Calif.). A portion of the pp65 pulsed and unpulsed hESC-DCswere X-ray irradiated at 2,000 rad for 4 minutes and 14 seconds usingthe Torrex 150D X-ray inspection system (EG&G Astrophysics ResearchCorporation, Long Beach, Calif.). The irradiated and non-irradiatedhESC-DCs were plated onto an ELISPOT plate at 1×10⁴ cells/100 ul/well ata 10:1 responder to stimulator ratio. The characterized PBMC responderswere thawed in 37° C. water bath, washed two times, resuspended in AIM-Vmedia (Invitrogen, Carlsbad, Calif.), and plated at 1×10⁵ cells/100ul/well on ELISPOT plates (Millipore, Bedford Mass.). ELISPOT plateswere coated with anti-IFNγ Ab (Mabtech, Mariemont, Ohio) at 10 ug/mlovernight (16-24 hours). The assay plates were placed at 37° C. and 5%CO₂ for 16-24 hours, and developed following the instructions providedby Mabtech. Spots were counted using a CTL Analyzer (Cellular TechnologyLimited, Decatur, Ill.). The results, shown in FIG. 10, demonstrateirradiated hESC-DCs maintained the capacity to stimulate IFNγ productionin an antigen specific manner.

Example 13 Chemotactic Migration of Irradiated mDC

Mature dendritic cells were differentiated according to the sameprotocol used in Example 12. The capacity of irradiated andnon-irradiated mDC (hESC-DCs) to migrate in the presence of thechemotactic ligand MIP3β (MIP3b in FIG. 11) using an in vitro transwellassay was investigated. AIM-V media (Invitrogen, Carlsbad, Calif.) wasadded to the upper and lower chambers of Transwell 24 well platescontaining 8.0 uM pore size inserts (Corning, Corning, N.Y.), andincubated overnight at 37° C., 5% CO₂ to equilibrate the membrane. mDCswere harvested and washed 2 times in AIM-V media. The cells wereresuspended in AIM-V media at 1.5×10⁶ cells/ml, and a portion of thesecells were X-ray irradiated at 2,000 rad using the Torrex 150D X-rayinspection system (EG&G Astrophysics Research Corporation, Long Beach,Calif.). After removal of the media from the transwell, 0.6 ml of AIM-Vwith or without the chemokine MIP3β (100 ng/ml) was added to the lowerchamber. A volume of 0.1 ml of irradiated or non-irradiated mDCs(0.15×10⁶ cells) was added to the top chamber. The transwell plate wasincubated for 2 hours at 37° C., 5% CO₂. The number of cells thatmigrated to the bottom chamber was determined using a hemacytometer. Theresults presented in FIG. 11 demonstrated irradiation did not affect theability of mDCs to migrate in response to MIP3β.

Example 14 Comparison of Cell Yield from Commercially Available Media

The effect on mDC cell yield of two commercially available media, mTeSR™serum free media (Stem Cell Technologies, Vancouver, BC, Canada) andXVIVO-10™ (Lonza, Walkersville, Md.) was investigated. H1 hESCs cultureand differentiation methods were performed as described in Example 1.The number of mature DCs at each harvest was compared between hESCs thatoriginated from XVIVO-10 or mTeSR cultures, FIG. 12. A total of 3harvests were performed from the initial differentiation. While bothmedia successfully produced mDC differentiated in vitro from hES, theresults suggest hESCs cultured in mTeSR provide better cell yields thanXVIVO-10.

Example 15 Maturation of hESC-Derived DCs Cultured in CommerciallyAvailable Media

Mature DCs possess the capacity to stimulate T cell responses; thereforeit is desirable to optimize the maturation process for hESC-derived DCs.(See, e.g. Chiara et al, 2007 We differentiated H1 hESCs as described inExample 1 but used Cellgro DC media during the steps to generateimmature and mature hESC-derived DC. After 24 hrs of maturation, cellswere harvested and evaluated for the presence of a mature DC phenotypebased on: 1) cell surface marker expression, 2) migration, and 3) IL-12expression.

Flow cytometry as described in Example 2 was used to analyze the cellsurface expression of DC associated markers: MHC class II, CD83, CD86,and CCR7. Mature hESC-derived DCs cultured in Cellgro™ DC media had bothelevated cell percent positive (%) and expression levels (MFI) of MHCclass II, CD83, and CCR7 compared to XVIVO-15™ cultured DCs, FIG. 13.The % of cells expressing CD86 levels remained unchanged, but the meanfluorescent intensity (MFI) was higher using Cellgro™ DC medium. Thisdata suggests Cellgro™ DC media promotes the generation of a more robustmature DC surface phenotype.

Next the migration efficiency of hESC-derived mature DCs cultured inCellgro™ DC and XVIVO-15™ media was studied using a Transwell assay asdescribed in Example 5. hESC-derived DCs cultured in XVIVO-15™ requiredto be matured for at least 48 hrs of maturation for effective migration.In contrast hESC-derived mature DCs cultured and matured in Cellgro™ DCmedia have an increased capacity to migrate in response to MIP3 betacompared to XVIVO-15™ at 24 hrs (FIG. 14). These data suggest thathESC-derived DCS cultured in Cellgro™ DC media have improved migrationat 24 hrs post maturation.

IL-12 helps promote a Th1 type immune response; therefore it would beuseful to optimize the expression of IL-12 from mature hESC-derived DCs.IL-12 expression levels were detected as described in Example 4.hESC-derived mature DCs cultured in Cellgro™ DC had higher IL-12expression levels (3.4 fold) compared to XVIVO-15™ cultured DCs, (FIG.15). The addition of IL-4 to the maturation cocktail can enhance IL-12expression from DCs (see, e.g. Ebner et al, (2001) J. Immunology166:633). IL-4 increased the expression of IL-12 from both mediumconditions, but hESC-derived mature DCs cultured in Cellgro™ DC mediahad markedly higher levels of IL-12 production (5.6 fold), (FIG. 15).Taken together, these data suggest hESC-derived mature DCs cultured inCellgro™ DC media have the capacity to express IL-12 at higher levelsthan DCs cultured in XVIVO-15™.

Example 16 Stimulation of 540 hTERT T Cell Lines by hESC-Derived DCsTransfected with RNA Encoding hTERT-LAMP

H1 hESCs were differentiated according to methods described in Example 1except mTeSR™ was used to culture hESCs and Cellgro™ DCs to generatehESC-derived immature and mature DCs. Between 2.0-4.0e6 hESC-derivedmature DCs were electroporated with RNA encoding hTERT-LAMP or GFP in0.4 cm cuvettes (Biorad, Hercules, Calif.) using a Biorad Gene PulserXcell (Biorad, Hercules, Calif.) using the following parameters: 300V,150 uF, and 100Ω. Electroporated cells were washed 1× in Cellgro™ DCmedia, and cells were transferred into maturation medium for anadditional 6 hrs. GFP- and hTERT-LAMP RNA electroporated hESC-derivedmature DCs were harvested and co-incubated with 540 hTERT T cell linesto detect the expression IFNγ as described in Examples 9 and 10. Resultsdemonstrated that hESC-derived mature DCs electroporated with hTERT-LAMPRNA stimulated increased levels of IFNγ from 540 hTERT specific T celllines compared to GFP-RNA transfected hESC-derived DCs, (FIG. 16). Thisdata suggests hESC-derived DCs have the capacity to process and presenthTERT antigen.

Many modifications and variations of this invention can be made withoutdeparting from its scope, as will be apparent to those skilled in theart. The specific embodiments described herein are offered by way ofexample only and are not meant to be limiting in any way. It is intendedthat the specification and examples be considered as exemplary only,with a true scope and spirit of the invention being indicated by thefollowing claims.

1-17. (canceled)
 18. A system for the production of antigen presentingcells comprising: a) a first isolated cell population comprising humanpluripotent stem cells in a cell culture medium comprisinggranulocyte-macrophage colony stimulating factor (GM-CSF) and bonemorphogenic protein 4 (BMP-4); and b) a second isolated cell populationcomprising mature dendritic cells which are the in vitro progeny of aportion of the first isolated cell population, wherein the system isstromal cell free.
 19. The system of claim 18, wherein at least 5% ofthe mature dendritic cells express one or more markers chosen from CD86and CD83.
 20. The system of claim 19, wherein the mature dendritic cellsexpressing one or more markers chosen from CD86 and CD83 further expressone or more of the following MHCII and CCR7.
 21. The system of claim 18,wherein the human pluripotent stem cells are human embryonic stem cells.22. The system of claim 18, wherein the system is serum free.
 23. Thesystem of claim 18, wherein the cell culture medium comprising GM-CSFand BMP-4 further comprises one or more factors selected from vascularendothelial growth factor (VEGF), stem cell factor (SCF), interleukin 4(IL-4) and interleukin 3 (IL-3).
 24. A cell culture, comprising: a) afirst isolated cell population comprising human pluripotent stem cellsin a cell culture medium comprising granulocyte-macrophage colonystimulating factor (GM-CSF) and bone morphogenic protein 4 (BMP-4) andb) a second isolated cell population comprising dendritic cells whichare the in vitro differentiated progeny of a portion of the firstisolated cell population, wherein the cell culture is stromal cell free.25. The cell culture of claim 24, wherein the human pluripotent stemcells are human embryonic stem cells.
 26. The cell culture of claim 24,wherein the cell culture is serum free.
 27. The cell culture of claim24, wherein the cell culture medium comprising GM-CSF and BMP-4 furthercomprises one or more factors selected from vascular endothelial growthfactor (VEGF), stem cell factor (SCF), interleukin 4 (IL-4) andinterleukin 3 (IL-3).
 28. A first and a second population of cells,comprising: a) a first isolated cell population comprising humanpluripotent stem cells in a cell culture medium comprisinggranulocyte-macrophage colony stimulating factor (GM-CSF) and bonemorphogenic protein 4 (BMP-4) and b) a second isolated cell populationcomprising dendritic cells which are the in vitro progeny of a portionof the first isolated cell population, wherein the first and the secondpopulation of cells are stromal cell free.
 29. The first and the secondpopulation of cells of claim 28, wherein the human pluripotent stemcells in the first isolated cell population are human embryonic stemcells.
 30. The first and the second population of cells of claim 28,wherein the first and the second populations are cultured in a serumfree medium.
 31. The first and the second population of cells of claim28, wherein the cell culture medium comprising GM-CSF and BMP-4 furthercomprises one or more factors selected from vascular endothelial growthfactor (VEGF), stem cell factor (SCF), interleukin 4 (IL-4) andinterleukin 3 (IL-3).